Generation of human steroidogenic cytochrome P450 enzymes for structural and functional characterization.

Six cytochrome P450 enzymes are involved in human steroidogenesis, converting cholesterol to sex steroids, mineralocorticoids, and glucocorticoids. While early work was accomplished with steroidogenic P450 orthologs from more accessible sources, knowledge of basic biochemistry through successful drug design have been greatly facilitated by recombinantly-expressed, highly purified human versions of these membrane proteins. Many membrane proteins are difficult to express and purify and are unstable. Membrane P450 expression in E. coli has been facilitated by modification and/or truncation of the membrane-interacting N-terminus, while metal-affinity resins and histidine-tagging greatly facilitates purification. However, substantial optimization is still frequently required to maintain protein stability. Over time, a generalized three-column purification scheme has been developed and tweaked to generate substantial quantities of fully active, highly purified human cytochrome P450 enzymes that have made possible the application of many structural, biochemical, and biophysical techniques to elucidate the mysteries of these critical human enzymes.

Selective steroidogenic cytochrome P450 haem iron ligation by steroid-derived isonitriles.

Alkyl isonitriles, R-NC, have previously been shown to ligate the heme (haem) iron of cytochromes P450 in both accessible oxidation states (ferrous, Fe2+, and ferric, Fe3+). Herein, the preparation of four steroid-derived isonitriles and their interactions with several P450s, including the steroidogenic CYP17A1 and CYP106A2, as well as the more promiscuous drug metabolizers CYP3A4 and CYP2D6, is described. It was found that successful ligation of the heme iron by the isonitrile functionality for a given P450 depends on both the position and stereochemistry of the isonitrile on the steroid skeleton. Spectral studies indicate that isonitrile ligation of the ferric heme is stable upon reduction to the ferrous form, with reoxidation resulting in the original complex. A crystallographic structure of CYP17A1 with an isonitrile derived from pregnanalone further confirmed the interaction and identified the absolute stereochemistry of the bound species.

Covariational reasoning and item context affect language in undergraduate mass balance written explanations.

Mass balance (MB) reasoning offers a rich topic for examination of students' scientific thinking and skills, as it requires students to account for multiple inputs and outputs within a system and apply covariational reasoning. Using previously validated constructed response prompts for MB, we examined 1,920 student-constructed responses (CRs) aligned to an emerging learning progression to determine how student language changes from low (1) to high (4) covariational reasoning levels. As students' abilities and thinking change with Context, we used the same general prompt in six physiological contexts. We asked how Level and Context affect student language and what language is conserved across Contexts at higher reasoning Levels. Using diversity methods, we found student language becomes more similar as covariational reasoning level increases. Using text analysis, we found context-dependent words at each Level; however, the type of context words changed. Specifically, at Level 1, students used context words that are tangential to MB reasoning, while Level 4 responses used words that specify inputs and outputs for the given Item Context. Further, at Level 4, students shared 30% of language across the six contexts and leveraged context-independent words including rate, equal, and some form of slower/lower/smaller. Together, these data demonstrate that Context affects undergraduate MB language at all covariational reasoning levels, but that the language becomes more specific and similar as Level increases. These findings encourage instructors to foster context-independent, comparative, and summative language during instruction to functionally build MB and covariational reasoning skills across contexts.NEW & NOTEWORTHY This article builds on the work of Scott et al. (Scott EE, Cerchiara J, McFarland JL, Wenderoth MP, Doherty JH. J Res Sci Teach 1: 37, 2023) and Shiroda et al. (Shiroda M, Fleming MP, Haudek KC. Front Educ 8: 989836, 2023) to quantitatively examine student language in written explanations of mass balance across six contexts using constructed response assessments. These results present an evaluation of student mass balance language and provide researchers and practitioners with tools to assist students in constructing scientific mass balance reasoning explanations.

Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate.

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6 Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

Updating the relationship of the Ne/ERN to task-related behavior: A brief review and suggestions for future research.

The error negativity/error-related negativity (Ne/ERN) is one of the most well-studied event-related potential (ERP) components in the electroencephalography (EEG) literature. Peaking about 50 ms after the commission of an error, the Ne/ERN is a negative deflection in the ERP waveform that is thought to reflect error processing in the brain. While its relationships to trait constructs such as anxiety are well-documented, there is still little known about how the Ne/ERN may subsequently influence task-related behavior. In other words, does the occurrence of the Ne/ERN trigger any sort of error corrective process, or any other behavioral adaptation to avoid errors? Several theories have emerged to explain how the Ne/ERN may implement or affect behavior on a task, but evidence supporting each has been mixed. In the following manuscript, we review these theories, and then systematically discuss the reasons that there may be discrepancies in the literature. We review both the inherent biological factors of the neural regions that underlie error-processing in the brain, and some of the researcher-induced factors in analytic and experimental choices that may be exacerbating these discrepancies. We end with a table of recommendations for future researchers who aim to understand the relationship between the Ne/ERN and behavior.

Measuring affect and complex working memory in natural and urban environments.

Introduction: Research suggests that spending time in natural environments is associated with cognitive and affective benefits, while increased use of technology and time spent in urban environments are associated with depletion of cognitive resources and an increasing prevalence of mental illness. Attention Restoration Theory suggests that exposure to natural environments can restore depleted attentional resources and thereby improve cognitive functioning and mood. Specifically, recent meta-analyses have revealed that the most improved cognitive abilities after nature exposure include selective attention, working memory, and cognitive flexibility. Methods: While existing studies examined these cognitive abilities, few have examined the Operation Span (OSPAN), a complex measure of working memory capacity. Therefore, the current study (N = 100) compared performance on the OSPAN and self-reported mood using the Positive and Negative Affect Schedule before and after a 30-min walk in a natural or urban environment. Results: Results from the study showed that both groups exhibited an increase in positive affect and a decrease in negative affect, suggesting that going outside for a walk can boost mood regardless of environment type. Inconsistent with past work, there were no significant changes in OSPAN scores before and after the walk for either environment type. Discussion: Future studies should analyze how the length of time spent in the environment, certain characteristics of the environment, and individual differences in connectedness to nature may impact attention restoration to gain insight on nature's ability to improve our affect and cognition.

What a Difference in Pressure Makes! A Framework Describing Undergraduate Students' Reasoning about Bulk Flow Down Pressure Gradients.

Pressure gradients serve as the key driving force for the bulk flow of fluids in biology (e.g., blood, air, phloem sap). However, students often struggle to understand the mechanism that causes these fluids to flow. To investigate student reasoning about bulk flow, we collected students' written responses to assessment items and interviewed students about their bulk flow ideas. From these data, we constructed a bulk flow pressure gradient reasoning framework that describes the different patterns in reasoning that students express about what causes fluids to flow and ordered those patterns into sequential levels from more informal ways of reasoning to more scientific, mechanistic ways of reasoning. We obtained validity evidence for this bulk flow pressure gradient reasoning framework by collecting and analyzing written responses from a national sample of undergraduate biology and allied health majors from 11 courses at five institutions. Instructors can use the bulk flow pressure gradient reasoning framework and assessment items to inform their instruction of this topic and formatively assess their students' progress toward more scientific, mechanistic ways of reasoning about this important physiological concept.

Oaks to arteries: the Physiology Core Concept of flow down gradients supports transfer of student reasoning.

The Physiology Core Concept of flow down gradients is a major concept in physiology, as pressure gradients are the key driving force for the bulk flow of fluids in biology. However, students struggle to understand that this principle is foundational to the mechanisms governing bulk flow across diverse physiological systems (e.g., blood flow, phloem sap flow). Our objective was to investigate whether bulk flow items that differ in scenario context (i.e., taxa, amount of scientific terminology, living or nonliving system) or in which aspect of the pressure gradient is kept constant (i.e., starting pressure or pressure gradient) influence undergraduate students' reasoning. Item scenario context did not impact the type of reasoning students used. However, students were more likely to use the Physiology Core Concept of "flow down [pressure] gradients" when the pressure gradient was kept constant and less likely to use this concept when the starting pressure was kept constant. We also investigated whether item scenario context or which aspect of the pressure gradient is kept constant impacted how consistent students were in the type of reasoning they used across two bulk flow items on the same homework. Most students were consistent across item scenario contexts (76%) and aspects of the pressure gradient kept constant (70%). Students who reasoned using "flow down gradients" on the first item were the most consistent (86, 89%), whereas students using "pressures indicate (but don't cause) flow" were the least consistent (43, 34%). Students who are less consistent know that pressure is somehow involved or indicates fluid flow but do not have a firm grasp of the concept of a pressure gradient as the driving force for fluid flow. These findings are the first empirical evidence to support the claim that using Physiology Core Concept reasoning supports transfer of knowledge across different physiological systems.NEW & NOTEWORTHY These findings are the first empirical evidence to support the claim that using Physiology Core Concept reasoning supports transfer of knowledge across different physiological systems.

Pyridine-containing substrate analogs are restricted from accessing the human cytochrome P450 8B1 active site by tryptophan 281.

The human oxysterol 12α-hydroxylase cytochrome P450 8B1 (CYP8B1) is a validated drug target for both type 2 diabetes and nonalcoholic fatty liver disease, but effective selective inhibitors are not yet available. Herein, steroidal substrate-mimicking compounds with a pyridine ring appended to the C12 site of metabolism were designed as inhibitors, synthesized, and evaluated in terms of their functional and structural interactions with CYP8B1. While the pyridine nitrogen was intended to coordinate the CYP8B1 active site heme iron, none of these compounds elicited shifts in the CYP8B1 Soret absorbance consistent with this type of interaction. However, when CYP8B1 was cocrystallized with the pyridine-containing compound with the 3-keto-Δ4 steroid backbone most similar to the endogenous substrate, it was apparent that this ligand was bound in a channel leading to the active site, instead of near the heme iron. Inspection of this structure suggested that tryptophan 281 directly above the heme might restrict active site binding of potential inhibitors with this design. This hypothesis was supported when a CYP8B1 W281F mutation did allow all three compounds to coordinate the heme iron as designed. These results indicated that the design of next-generation CYP8B1 inhibitors should be compatible with the low-ceiling tryptophan immediately above the heme iron.

Human cytochrome P450 17A1 structures with metabolites of prostate cancer drug abiraterone reveal substrate-binding plasticity and a second binding site.

Abiraterone acetate is a first-line therapy for castration-resistant prostate cancer. This prodrug is deacetylated in vivo to abiraterone, which is a potent and specific inhibitor of cytochrome P450 17A1 (CYP17A1). CYP17A1 performs two sequential steps that are required for the biosynthesis of androgens that drive prostate cancer proliferation, analogous to estrogens in breast cancer. Abiraterone can be further metabolized in vivo on the steroid A ring to multiple metabolites that also inhibit CYP17A1. Despite its design as an active-site-directed substrate analog, abiraterone and its metabolites demonstrate mixed competitive/noncompetitive inhibition. To understand their binding, we solved the X-ray structures of CYP17A1 with three primary abiraterone metabolites. Despite different conformations of the steroid A ring and substituents, all three bound in the CYP17A1 active site with the steroid core packed against the I helix and the A ring C3 keto or hydroxyl oxygen forming a hydrogen bond with N202 similar to abiraterone itself. The structure of CYP17A1 with 3-keto, 5α-abiraterone was solved to 2.0 Å, the highest resolution to date for a CYP17A1 complex. This structure had additional electron density near the F/G loop, which is likely a second molecule of the inhibitor and which may explain the noncompetitive inhibition. Mutation of the adjacent Asn52 to Tyr positions its side chain in this space, maintains enzyme activity, and prevents binding of the peripheral ligand. Collectively, our findings provide further insight into abiraterone metabolite binding and CYP17A1 function.

Four Decades of Cytochrome P450 2B Research: From Protein Adducts to Protein Structures and Beyond.

This article features selected findings from the senior author and colleagues dating back to 1978 and covering approximately three-fourths of the 60 years since the discovery of cytochrome P450. Considering the vast number of P450 enzymes in this amazing superfamily and their importance for so many fields of science and medicine, including drug design and development, drug therapy, environmental health, and biotechnology, a comprehensive review of even a single topic is daunting. To make a meaningful contribution to the 50th anniversary of Drug Metabolism and Disposition, we trace the development of the research in a single P450 laboratory through the eyes of seven individuals with different backgrounds, perspectives, and subsequent career trajectories. All co-authors are united in their fascination for the structural basis of mammalian P450 substrate and inhibitor selectivity and using such information to improve drug design and therapy. An underlying theme is how technological advances enable scientific discoveries that were impossible and even inconceivable to prior generations. The work performed spans the continuum from: 1) purification of P450 enzymes from animal tissues to purification of expressed human P450 enzymes and their site-directed mutants from bacteria; 2) inhibition, metabolism, and spectral studies to isothermal titration calorimetry, deuterium exchange mass spectrometry, and NMR; 3) homology models based on bacterial P450 X-ray crystal structures to rabbit and human P450 structures in complex with a wide variety of ligands. Our hope is that humanizing the scientific endeavor will encourage new generations of scientists to make fundamental new discoveries in the P450 field. SIGNIFICANCE STATEMENT: The manuscript summarizes four decades of work from Dr. James Halpert's laboratory, whose investigations have shaped the cytochrome P450 field, and provides insightful perspectives of the co-authors. This work will also inspire future drug metabolism scientists to make critical new discoveries in the cytochrome P450 field.

Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition.

Human cytochrome P450 11B1 (CYP11B1) generation of the major glucocorticoid cortisol requires two electrons delivered sequentially by the iron­sulfur protein adrenodoxin. While the expected adrenodoxin binding site is on the opposite side of the heme and 15-20 Å away, evidence is provided that adrenodoxin allosterically impacts CYP11B1 ligand binding and catalysis. The presence of adrenodoxin both decreases the dissociation constant (Kd) for substrate binding and increases the proportion of substrate that is bound at saturation. Adrenodoxin additionally decreases the Michaelis-Menten constant for the native substrate. Similar studies with several inhibitors also demonstrate the ability of adrenodoxin to modulate inhibition (IC50 values). Somewhat similar allosterism has recently been observed for the closely related CYP11B2/aldosterone synthase, but there are several marked differences in adrenodoxin effects on the two CYP11B enzymes. Comparison of the sequences and structures of these two CYP11B enzymes helps identify regions likely responsible for the functional differences. The allosteric effects of adrenodoxin on CYP11B enzymes underscore the importance of considering P450/redox partner interactions when evaluating new inhibitors.

The structure and characterization of human cytochrome P450 8B1 supports future drug design for nonalcoholic fatty liver disease and diabetes.

Human cytochrome P450 8B1 (CYP8B1) is involved in conversion of cholesterol to bile acids. It hydroxylates the steroid ring at C12 to ultimately produce the bile acid cholic acid. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. Herein, the human CYP8B1 protein was generated and used to identify and characterize interactions with a series of azole inhibitors, which tend to be poorly selective P450 inhibitors. Structurally related miconazole, econazole, and tioconazole bound with submicromolar dissociation constants and were effective inhibitors of the native reaction. CYP8B was cocrystallized with S-tioconazole to yield the first X-ray structure. This inhibitor bound in the active site with its azole nitrogen coordinating the heme iron, consistent with inhibitor binding and inhibition assay data. Additionally, the CYP8B1 active site was compared with similar P450 enzymes to identify features that may facilitate the design of more selective inhibitors. Selective inhibitors should promote a better understanding of the role of CYP8B1 inhibition in normal physiology and disease states and provide a possible treatment for nonalcoholic fatty liver disease and type 2 diabetes.

Cytochrome P450 Binding and Bioactivation of Tumor-Targeted Duocarmycin Agents.

Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.

Effects of fluorine substitution on substrate conversion by cytochromes P450 17A1 and 21A2.

Cytochromes P450 17A1 (CYP7A1) and 21A2 (CYP21A2) catalyze key reactions in the production of steroid hormones, including mineralocorticoids, glucocorticoids, and androgens. With the ultimate goal of designing probes that are selectively metabolized to each of these steroid types, fluorinated derivatives of the endogenous substrates, pregnenolone and progesterone, were prepared to study the effects on CYP17A1 and CYP21A2 activity. In the functional assays, the hydroxylase reactions catalysed by each of these enzymes were blocked when fluorine was introduced at the site of metabolism (positions 17 and 21 of the steroid core, respectively). CYP17A1, furthermore, performed the 17,20-lyase reaction on substrates with a fluorine installed at the 21-position. Importantly, none of the substitutions examined herein prevented compound entry into the active sites of either CYP17A1 or CYP21A2 as demonstrated by spectral binding assays. Taken together, the results suggest that fluorine might be used to redirect the metabolic pathways of pregnenolone and progesterone to specific types of steroids.

Aldosterone Synthase Structure With Cushing Disease Drug LCI699 Highlights Avenues for Selective CYP11B Drug Design.

[Figure: see text].

Structural and functional insights into aldosterone synthase interaction with its redox partner protein adrenodoxin.

Aldosterone is the major mineralocorticoid in the human body controlling blood pressure and salt homeostasis. Overproduction of aldosterone leads to primary aldosteronism, which is the most common form of secondary hypertension with limited treatment options. Production of aldosterone by cytochrome P450 11B2 (CYP11B2, aldosterone synthase) requires two reduction events with the electrons delivered by the iron/sulfur protein adrenodoxin. Very limited information is available about the structural and functional basis of adrenodoxin/CYP11B2 interaction, which impedes the development of new treatment options for primary aldosteronism. A systematic study was carried out to determine if adrenodoxin interaction with CYP11B2 might also have an allosteric component in addition to electron transfer. Indeed, local increases in adrenodoxin concentration promote binding of the substrate 11-deoxycorticosterone and the inhibitor osilodrostat (LCI699) in the active site-over 17 Å away-as well as enhance the inhibitory effect of this latter drug. The CYP11B2 structure in complex with adrenodoxin identified specific residues at the protein-protein interface interacting via five salt bridges and four hydrogen bonds. Comparisons with cholesterol-metabolizing CYP11A1 and cortisol-producing CYP11B1, which also bind adrenodoxin, revealed substantial structural differences in these regions. The structural and functional differences between different P450 interactions with adrenodoxin may provide valuable clues for an orthogonal treatment approach for primary aldosteronism by specifically targeting the interaction between CYP11B2 and adrenodoxin.

Steroidogenic cytochrome P450 17A1 structure and function.

Cytochrome P450 17A1 (CYP17A1) is a critical steroidogenic enzyme, essential for producing glucocorticoids and sex hormones. This review discusses the complex activity of CYP17A1, looking at its role in both the classical and backdoor steroidogenic pathways and the complex chemistry it carries out to perform both a hydroxylation reaction and a carbon-carbon cleavage, or lyase reaction. Functional and structural investigations have informed our knowledge of these two reactions. This review focuses on a few specific aspects of this discussion: the identities of reaction intermediates, the coordination of hydroxylation and lyase reactions, the effects of cytochrome b5, and conformational selection. These discussions improve understanding of CYP17A1 in a physiological setting, where CYP17A1 is implicated in a variety of steroidogenic diseases. This information can be used to improve ways in which CYP17A1 can be effectively modulated to treat diseases such as prostate and breast cancer, Cushing's syndrome, and glioblastoma.