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Theoretical Studies of Drug-Metabolizing Cytochrome P450 Enzymes
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The family of cytochrome P450 enzymes (P450s) belongs to one of the most important enzyme families in the human body. P450s are involved in the synthesis of endogenous compounds and metabolism of exogenous substances. In mammalian species, drug metabolizing P450s are anchored in the bilayer lipid membrane, which allows the enzymes to interact with other proteins and ligand molecules. A wealth of knowledge about the structures, functions, and mechanisms of P450s have been obtained from both experimental and theoretical studies. However, the mechanisms behind some experimental results, such as the regio- and stereoselectivity and structural flexibility are still elusive.

In this thesis, I present the work done in my doctoral studies, which was focused on the catalytic selectivity and structural flexibility of P450s. Multiple theoretical modeling approaches, such as homology modeling, molecular docking, molecular dynamics, quantum mechanics, and quantum mechanics/molecular mechanics, were applied in the studies. In papers I and II, the regio- and stereoselectivity of CYP4F2, CYP3A4, and CYP19A1 catalyzed C–H hydroxylation of different substrates were studied. The results indicate that the ligand reactivity and accessibility can be decisive for the regio- and stereoselectivity. However, which of them is more important is system-dependent. The quantum mechanics/molecular mechanics calculation results imply that the distribution of spin natural orbitals could be used for discriminating the roles of the reactivity and accessibility. In papers III and IV, the conformational dynamics of the open and closed structures of CYP2B4 and the ligand cooperativity phenomenon of midazolam metabolized by CYP3A4 were investigated using molecular dynamics simulations. From the simulation results, we identified the key residues for the conformational dynamics for the open-to-intermediate transition and found that the ligand cooperativity is also caused by the large flexibility of P450. The results also indicated that the homotropic cooperativity mainly occurs in the large and flexible productive site, rather than in the remote allosteric site.

Abstract [sv]

Familjen av cytokrom P450-enzymer (P450) tillhör en av de viktigaste enzymfamiljerna i människokroppen. P450 är involverade i syntesen av endogena föreningar och metabolism av exogena substanser. Hos däggdjursarter är läkemedelsmetaboliserande P450s bundna till lipidmembranet i cellerna, vilket påverkar P450s förmåga att interagera med andra proteiner och ligandmolekyler. En mängd kunskap om P450:s struktur, funktion och mekanism har erhållits från både experimentella och teoretiska studier. Däremot är mekanismerna bakom vissa experimentella resultat, såsom regio- och stereoselektivitet och strukturell flexibilitet fortfarande svårfångade.

 

I denna avhandling presenterar jag det arbete som gjorts under mina doktorandstudier, som fokuserade på den katalytiska selektiviteten och strukturella flexibiliteten hos P450. Flera teoretiska modelleringsmetoder, såsom homologimodellering, molekylär dockning, molekyldynamik, kvantmekanik och kvantmekanik/molekylmekanik har använts i studierna. I artikel I och II studerades regio- och stereoselektiviteten för CYP4F2, CYP3A4 och CYP19A1 C-H hydroxylering av olika substrat. Resultaten indikerar att ligandreaktiviteten och tillgängligheten kan vara avgörande för regio- och stereoselektiviteten. Vilken av dem som är viktigare är emellertid systemberoende. Resultaten av beräkningen med kvantmekanik/molekylmekanik innebär att fördelningen av naturliga spin orbitaler kan användas för att urskilja rollen för substratets reaktivitet och tillgänglighet. I artiklarna III och IV undersöktes konformationsdynamiken för de öppna och slutna strukturerna av CYP2B4 och ligandkooperativitetsfenomenet för midazolam metaboliserat genom CYP3A4 med hjälp av molekyldynamiksimuleringar. Från simuleringsresultaten identifierade vi nyckelaminosyrorna för konformationens dynamik för den öppen-till-intermediär-övergången och fann att ligandens kooperativitet också orsakas av den stora flexibiliteten hos P450. Resultaten indikerade även att den homotropa kooperativiteten huvudsakligen inträffar på det stora och flexibla produktiva sätet, snarare än på det avlägsna allosteriska sätet.

Place, publisher, year, edition, pages
Stockholm, Sweden: KTH Royal Institute of Technology, 2020. , p. 75
Series
TRITA-CBH-FOU ; 2020:29
Keywords [en]
Cytochrome P450 enzymes, molecular dynamics, quantum chemistry, structural flexibililty, ONIOM
National Category
Natural Sciences
Research subject
Theoretical Chemistry and Biology
Identifiers
URN: urn:nbn:se:kth:diva-273361ISBN: 978-91-7873-546-4 (print)OAI: oai:DiVA.org:kth-273361DiVA, id: diva2:1430348
Public defence
2020-06-10, https://kth-se.zoom.us/webinar/register/WN_cYqhoHWeSuCidzUAeMPKYg, 10:00 (English)
Opponent
Supervisors
Note

QC 2020-05-20

Available from: 2020-05-20 Created: 2020-05-14 Last updated: 2020-05-28Bibliographically approved
List of papers
1. Computational Insight Into Vitamin K-1 omega-Hydroxylation by Cytochrome P450 4F2
Open this publication in new window or tab >>Computational Insight Into Vitamin K-1 omega-Hydroxylation by Cytochrome P450 4F2
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2018 (English)In: Frontiers in Pharmacology, ISSN 1663-9812, E-ISSN 1663-9812, Vol. 9, article id 1065Article in journal (Refereed) Published
Abstract [en]

Vitamin K-1 (VK1) plays an important role in the modulation of bleeding disorders. It has been reported that omega-hydroxylation on the VK1 aliphatic chain is catalyzed by cytochrome P450 4F2 (CYP4F2), an enzyme responsible for the metabolism of eicosanoids. However, the mechanism of VK1 omega-hydroxylation by CYP4F2 has not been disclosed. In this study, we employed a combination of quantum mechanism (QM) calculations, homology modeling, molecular docking, molecular dynamics (MD) simulations, and combined quantum mechanism/molecular mechanism (QM/MM) calculations to investigate the metabolism profile of VK1 omega-hydroxylation. QM calculations based on the truncated VK1 model show that the energy barrier for omega-hydroxylation is about 6-25 kJ/mol higher than those at other potential sites of metabolism. However, results from the MD simulations indicate that hydroxylation at the omega-site is more favorable than at the other potential sites, which is in accordance with the experimental observation. The evaluation of MD simulations was further endorsed by the QM/MM calculation results. Our studies thus suggest that the active site residues of CYP4F2 play a determinant role in the omega-hydroxylation. Our results provide structural insights into the mechanism of VK1 omega-hydroxylation by CYP4F2 at the atomistic level and are helpful not only for characterizing the CYP4F2 functions but also for looking into the omega-hydroxylation mediated by other CYP4 enzymes.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
cytochrome P450, CYP4F2, omega-hydroxylation, molecular dynamics, QM/MM, homology modeling, vitamin K-1
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:kth:diva-235873 (URN)10.3389/fphar.2018.01065 (DOI)000445589400001 ()30319412 (PubMedID)2-s2.0-85055145671 (Scopus ID)
Note

QC 20181009

Available from: 2018-10-09 Created: 2018-10-09 Last updated: 2020-05-23Bibliographically approved
2. Mechanistic Insights into the Regio‐ and Stereoselectivities of Testosterone and Dihydrotestosterone Hydroxylation Catalyzed by CYP3A4 and CYP19A1
Open this publication in new window or tab >>Mechanistic Insights into the Regio‐ and Stereoselectivities of Testosterone and Dihydrotestosterone Hydroxylation Catalyzed by CYP3A4 and CYP19A1
2020 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 26, p. 6214-6223Article in journal (Refereed) In press
Abstract [en]

The hydroxylation of nonreactive C−H bonds can be easily catalyzed by a variety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically. However, understanding the regio‐ and stereoselectivities of substrates hydroxylated by P450s remains a great challenge. Herein, we use a multi‐scale modeling approach to investigate the selectivity of testosterone (TES) and dihydrotestosterone (DHT) hydroxylation catalyzed by two important P450s, CYP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHT were predicted by dockings and molecular dynamics simulations, in which the experimentally identified sites of metabolism of TES/DHT can access to the catalytic center. The regio‐ and stereoselectivities of TES/DHT hydroxylation were further evaluated by quantum mechanical and ONIOM calculations. For CYP19A1, we found that sites 1β, 2β and 19 can access the catalytic center, with the intrinsic reactivity 2β>1β>19. However, our ONIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT, which is consistent with the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlying the selectivity of TES/DHT hydroxylation mediated by CYP3A4 and CYP19A1 and is helpful for understanding the selectivity of other substrates that are hydroxylated by P450s.

Place, publisher, year, edition, pages
Weinheim, Germany: , 2020
Keywords
C-H hydroxylation, density functional calculations, hydroxylation, molecular modeling, P450, steroids
National Category
Theoretical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-273352 (URN)10.1002/chem.201905272 (DOI)000529008500001 ()32049373 (PubMedID)2-s2.0-85083973024 (Scopus ID)
Projects
Biology
Funder
Swedish National Infrastructure for Computing (SNIC), SNIC-2019-3-636
Note

QC 20200523

Available from: 2020-05-14 Created: 2020-05-14 Last updated: 2020-05-23Bibliographically approved
3. Dissecting the Structural Plasticity and Dynamics of Cytochrome P450 2B4 by Molecular Dynamics Simulations
Open this publication in new window or tab >>Dissecting the Structural Plasticity and Dynamics of Cytochrome P450 2B4 by Molecular Dynamics Simulations
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The plasticity of cytochrome P450 enzymes (P450s) is known to contribute significantly to their catalytic capacity of metabolizing various substrates. Although numerous studies have been performed, factors governing the plasticity and dynamics of P450s are still not fully understood. In this study, taking CYP2B4 as an example, we dissect the protein plasticity and dynamics in different environments. CYP2B4 is featured by a high degree of plasticity that exhibits open, closed, and intermediate states. By analyzing the CYP2B4 crystal structures, we identified the structural features for the closed, open and intermediate states. Interestingly, formation of the dimer structure was found in the open and intermediate structures. The subsequent MD simulations of the open structure in water confirmed the importance of the dimer form in stabilizing the open conformations. MD simulations of the closed and open structures in the membrane environment and the free energies for opening the F-G cassette obtained from the umbrella sampling calculations indicate that the membrane environment is important for stabilizing the F-G cassette. The dynamical network analysis indicates that Asp105 on the B-C loop plays an important role in transiting the structure from the open to intermediate. Our results thus unveil the mechanism of dimer formation and open-to-intermediate transition for CYP2B4 in the water and membrane environments.

Keywords
P450, Conformational plasticity, Molecular dynamics, CYP2B4, Umbrella sampling
National Category
Theoretical Chemistry Other Biological Topics
Identifiers
urn:nbn:se:kth:diva-273358 (URN)
Note

QC 20200518

Available from: 2020-05-14 Created: 2020-05-14 Last updated: 2020-05-18Bibliographically approved
4. Mechanism of the Homotropic Cooperativity of Midazolam Metabolism by Cytochrome P450 3A4: Insight from Computational Studies
Open this publication in new window or tab >>Mechanism of the Homotropic Cooperativity of Midazolam Metabolism by Cytochrome P450 3A4: Insight from Computational Studies
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Midazolam (MDZ) is a commonly used drug and is metabolized by cytochrome P450 3A4 (CYP3A4). It has been reported that the ratio of the hydroxylation products, 1'-OH-MDZ/4-OH-MDZ, is dependent on the MDZ concentration, which reflects that there exists the homotropic cooperative behavior in the CYP3A4-mediated hydroxylation of MDZ. Here, we used quantum chemistry (QC), molecular docking, conventional molecular dynamics (cMD) simulation, and Gaussian accelerated molecular dynamics (GaMD) simulation approaches to investigate the mechanism of the interactions between CYP3A4 and MDZ. Our study suggests that the H41 site, i.e. the pro-R center, is the most reactive site for the hydrogen abstraction, followed by the C1' site. However, the product 4-OH-MDZ is likely to be racemic due to the chirality inversion in the rebound step. We found that the allosteric site was not involved in the ligand cooperativity and the observation that there exists one or two MDZs in the productive site is in line with the experimental observations.

Keywords
CYP3A4, homotropic cooperativity, midazolam, quantum chemistry, molecular dynamics
National Category
Other Biological Topics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-273359 (URN)
Projects
Chemistry
Note

QC 20200518

Available from: 2020-05-14 Created: 2020-05-14 Last updated: 2020-05-18Bibliographically approved

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The full text will be freely available from 2020-06-15 15:00
Available from 2020-06-15 15:00

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