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1.
Methods Mol Biol ; 2266: 105-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33759123

RESUMO

Interactions between enzymes and small molecules lie in the center of many fundamental biochemical processes. Their analysis using molecular dynamics simulations have high computational demands, geometric approaches fail to consider chemical forces, and molecular docking offers only static information. Recently, we proposed to combine molecular docking and geometric approaches in an application called CaverDock. CaverDock is discretizing enzyme tunnel into discs, iteratively docking with restraints into one disc after another and searching for a trajectory of the ligand passing through the tunnel. Here, we focus on the practical side of its usage describing the whole method: from getting the application, and processing the data through a workflow, to interpreting the results. Moreover, we shared the best practices, recommended how to solve the most common issues, and demonstrated its application on three use cases.


Assuntos
Descoberta de Drogas/métodos , Simulação de Acoplamento Molecular/métodos , Proteínas/química , Ácido Araquidônico/química , Sítios de Ligação , Cloridrinas/química , Sistema Enzimático do Citocromo P-450/química , Desenho de Fármacos , Etanol/análogos & derivados , Etanol/química , Dibrometo de Etileno/química , Hidrolases/química , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Software , Relação Estrutura-Atividade , Termodinâmica
2.
Nat Commun ; 12(1): 1621, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712579

RESUMO

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and ß-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Simulação de Dinâmica Molecular , Mutação , Catálise , Domínio Catalítico/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Cinética , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por Substrato
3.
Arch Biochem Biophys ; 698: 108732, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33358998

RESUMO

The ubiquitous flavoenzymes commonly catalyze redox chemistry such as the monooxygenation of organic substrates and are both widely utilized in nature (e.g., in primary and secondary metabolism) and of significant industrial interest. In this work, we highlight the structural and mechanistic characteristics of the distinct types of flavoprotein monooxygenases (FPMOs). We thereby illustrate the chemical basis of FPMO catalysis, which enables reactions such as (aromatic) hydroxylation, epoxidation, (de)halogenation, heteroatom oxygenation, Baeyer-Villiger oxidation, α-hydroxylation of ketones, or non-oxidative carbon-hetero bond cleavage. This seemingly unmatched versatility in oxygenation chemistry results from extensive fine-tuning and regiospecific functionalization of the flavin cofactor that is tightly controlled by the surrounding protein matrix. Accordingly, FPMOs steer the formation of covalent flavin-oxygen adducts for oxygen transfer in the form of the classical flavin-C4a-(hydro)peroxide or the recently discovered N5-functionalized flavins (i.e. the flavin-N5-oxide and the flavin-N5-peroxide), while in rare cases covalent oxygen adduct formation may be foregone entirely. Finally, we speculate about hitherto undiscovered flavin-mediated oxygenation reactions and compare FPMOs to cytochrome P450 monooxygenases, before addressing open questions and challenges for the future investigation of FPMOs.


Assuntos
Flavoproteínas/química , Oxigenases de Função Mista/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Biocatálise , Sistema Enzimático do Citocromo P-450/química , Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Oxigenases de Função Mista/classificação , Modelos Químicos , Oxigênio/química
4.
Chem Commun (Camb) ; 57(4): 520-523, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33331834

RESUMO

Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.


Assuntos
Antidepressivos/síntese química , Sistema Enzimático do Citocromo P-450/química , Ketamina/análogos & derivados , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Hidroxilação , Ketamina/síntese química , Ketamina/química , Ketamina/metabolismo , Simulação de Acoplamento Molecular , Mutação , Oxirredução , Ligação Proteica , Streptomyces coelicolor/enzimologia , /enzimologia
5.
J Med Chem ; 63(24): 15752-15772, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33307675

RESUMO

ADP-mediated platelet aggregation is signaled through G protein-coupled receptors P2Y1 and P2Y12 on the platelet. The clinical effectiveness of inhibiting P2Y12 has been well established, and preclinical studies indicated that the inhibition of P2Y1 could provide equivalent antithrombotic efficacy as P2Y12 antagonists and reduce bleeding risks. On the basis of the 2-phenyl-1H-imidazole scaffold of our previously reported xanthine oxidase inhibitor WSJ-557, we first achieved the transition from the xanthine oxidase inhibitors to dual-target antagonists against P2Y1 and P2Y12. We described the structure-activity relationships of the 2-phenyl-1H-imidazole compounds, which led to the identification of the most potent antiplatelet agents, 24w and 25w, both showing a rapid onset of action in pharmacokinetic study. Furthermore, the rat model suggested that 24w demonstrated a wider therapeutic window than ticagrelor, displaying equivalent and dose-dependent antithrombotic efficacy with lower blood loss compared to ticagrelor at same oral dose. These results supported that 24w and 25w could be promising drug candidates.


Assuntos
Inibidores Enzimáticos/química , Inibidores da Agregação de Plaquetas/química , Antagonistas do Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y1/química , Xantina Oxidase/antagonistas & inibidores , Animais , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Estabilidade de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Meia-Vida , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/metabolismo , Inibidores da Agregação de Plaquetas/farmacologia , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Ratos , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Relação Estrutura-Atividade , Ticagrelor/farmacologia , Xantina Oxidase/metabolismo
6.
J Comput Aided Mol Des ; 34(12): 1237-1259, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33034007

RESUMO

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/efeitos dos fármacos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
7.
Plant Mol Biol ; 104(3): 327-337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761540

RESUMO

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.


Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Transcriptoma
8.
J Chem Inf Model ; 60(10): 5255-5264, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-32846088

RESUMO

The surface of proteins is vital in determining protein functions. Herein, a program, Protein Surface Printer (PSP), is built that performs multiple functions in quantifying protein surface domains. Two proteins, PETase and cytochrome P450, are used to validate that the program supports atomistic simulations with different combinations of programs and force fields. A case study is conducted on the structural analysis of the spike proteins of SARS-CoV-2 and SARS-CoV and the human cell receptor ACE2. Although the surface domains of both spike proteins are highly similar, their receptor-binding domains (RBDs) and the O-linked glycan domains are structurally different. The O-linked glycan domain of SARS-CoV-2 is highly positively charged, which may promote binding to negatively charged human cells.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/metabolismo , Vírus da SARS/metabolismo , Software , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/química , Betacoronavirus/fisiologia , Sítios de Ligação , Infecções por Coronavirus/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/metabolismo , Ligação Proteica , Domínios Proteicos , Vírus da SARS/química , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/metabolismo , Glicoproteína da Espícula de Coronavírus/química
9.
Arch Biochem Biophys ; 692: 108544, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32822639

RESUMO

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 µM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.


Assuntos
Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes , Sirolimo/metabolismo , Streptomyces/genética
10.
Chem Biol Interact ; 329: 109147, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32738202

RESUMO

Acacetin is a natural flavonoid that is widely distributed in plants and possesses numerous pharmacological activities. The aim of the present study was to investigate the effects of acacetin on the activities of the cytochrome P450 family members CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1, and CYP3A2 in rat liver microsomes in vitro and rats in vivo to evaluate potential herb-drug interactions by using a cocktail approach. Phenacetin, bupropion, tolbutamide, dextromethorphan, chlorzoxazone, and midazolam were chosen as the probe substrates. An ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the probe substrates and their metabolites. In vitro, the mode of acacetin inhibition of CYP2B1, CYP2C11, and CYP2E1 was competitive, while mixed inhibition was observed for CYP1A2 and CYP3A2. The Ki values in this study were less than 8.32 µM. In vivo, the mixed probe substrates were administered by gavage after daily intraperitoneal injection with 50 mg/kg acacetin or saline for 2 weeks. The main pharmacokinetic parameters, area under the plasma concentration-time curve (AUC), plasma clearance (CL), and maximum plasma concentration (Cmax) of the probe substrates were significantly different in the experimental group than in the control group. Overall, the in vitro and in vivo results indicated that acacetin would be at high risk to cause toxicity and drug interactions via cytochrome P450 inhibition.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/metabolismo , Animais , Área Sob a Curva , Sistema Enzimático do Citocromo P-450/química , Flavonas/química , Flavonas/farmacocinética , Meia-Vida , Concentração Inibidora 50 , Cinética , Masculino , Microssomos Hepáticos/metabolismo , Curva ROC , Ratos , Ratos Sprague-Dawley
11.
J Med Chem ; 63(15): 8059-8068, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32643929

RESUMO

Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis. Idiosyncratic drug reactions, due to clinical application of pirfenidone, have been documented, even along with death cases resulting from acute liver failure. The present study aimed at the investigation of metabolic activation of pirfenidone possibly participating in the reported adverse reactions. Pirfenidone-derived glutathione/N-acetylcysteine (GSH/NAC) conjugates were detected in microsomal/primary hepatocyte incubations after exposure to pirfenidone. The GSH/NAC conjugates were also observed in bile and urine of rats given pirfenidone, respectively. The observation of the conjugates suggests the formation of a quinone methide intermediate derived from pirfenidone. The intermediate was possibly generated through two pathways. First, pirfenidone was directly metabolized to the quinone methide intermediate via dehydrogenation; second, pirfenidone was oxidized to 5-hydroxymethyl pirfenidone, followed by sulfation to a benzyl alcohol-sulfate derivative. The findings facilitate the understanding of the mechanisms of pirfenidone-induced idiosyncratic toxicity and assist medicinal chemists to minimize toxicities in the development of new pharmaceutical agents.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Piridonas/metabolismo , Sulfotransferases/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Sistema Enzimático do Citocromo P-450/química , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Piridonas/química , Piridonas/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sulfotransferases/química
12.
J Med Chem ; 63(11): 5865-5878, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32390424

RESUMO

Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.


Assuntos
Anticonvulsivantes/uso terapêutico , Convulsões/tratamento farmacológico , Vitamina K/análogos & derivados , Administração Oral , Animais , Anticonvulsivantes/química , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/farmacologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Convulsões/patologia , Relação Estrutura-Atividade , Vitamina K/farmacocinética , Vitamina K/farmacologia , Vitamina K/uso terapêutico , Peixe-Zebra
13.
Sci Rep ; 10(1): 7284, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350331

RESUMO

The simulation of membrane proteins requires compatible protein and lipid force fields that reproduce the properties of both the protein and the lipid bilayer. Cytochrome P450 enzymes are bitopic membrane proteins with a transmembrane helical anchor and a large cytosolic globular domain that dips into the membrane. As such, they are representative and challenging examples of membrane proteins for simulations, displaying features of both peripheral and integral membrane proteins. We performed molecular dynamics simulations of three cytochrome P450 isoforms (2C9, 2C19 and 1A1) in a 2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphocholine bilayer using two AMBER force field combinations: GAFF-LIPID with ff99SB for the protein, and LIPID14 with ff14SB for the protein. Comparison of the structural and dynamic properties of the proteins, the lipids and the protein-membrane interactions shows differing sensitivity of the cytochrome P450 isoforms to the choice of force field, with generally better agreement with experiment for the LIPID14 + ff14SB combination.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Animais , Humanos , Estrutura Secundária de Proteína
14.
PLoS One ; 15(5): e0231980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357188

RESUMO

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de Sequência
15.
Inorg Chem ; 59(12): 8034-8043, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32452669

RESUMO

Cytochrome (Cyt) P450s are an important class of enzymes with numerous functions in nature. The unique reactivity of these enzymes relates to their heme b active sites with an axially bound, deprotonated cysteine (a "cysteinate") ligand (chemically speaking a thiolate). The heme-thiolate active sites further contain a number of conserved hydrogen-bonds (H-bonds) to the bound cysteinate ligand, which have been proposed to tune and stabilize the Fe-S bond. In this work, we present the low-temperature preparation of five ferric heme-thiolate nitric oxide (NO) model complexes that contain one tunable hydrogen-bond to the bound thiolate ligand. We show that the presence of a H-bond has a dramatic effect in stabilizing the thiolate ligand against direct reaction with NO. This observation reinforces the important protective role of H-bonds in Cyt P450s. We further demonstrate that H-bond strength tunes thiolate donor strength, which, in turn, controls the N-O and Fe-NO stretching frequencies and hence, bond strengths. We observe a direct correlation between the Fe-NO and N-O stretching frequencies, indicative of a thiolate σ-trans effect (interaction). Here, very small changes in H-bond strength lead to a surprisingly large effect on the FeNO unit. This result implies that subtle changes in the Cys-pocket of a Cyt P450 can strongly affect reactivity. Importantly, using the Fe-NO/N-O correlation established here, the thiolate donor strength in heme-thiolate enzyme active sites and model complexes can be quantified in a straightforward way, using NO as a probe. This spectroscopic correlation provides a quantitative measure of the thiolate's "push" effect, which is important in O2-activation (Compound I formation) in Cyt P450s in general.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Compostos Férricos/química , Modelos Químicos , Compostos de Sulfidrila/química , Sistema Enzimático do Citocromo P-450/metabolismo , Teoria da Densidade Funcional , Compostos Férricos/metabolismo , Ligação de Hidrogênio , Compostos de Sulfidrila/metabolismo
16.
Nucleic Acids Res ; 48(W1): W580-W585, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32182358

RESUMO

Cytochrome P450 enzymes (CYPs)-mediated drug metabolism influences drug pharmacokinetics and results in adverse outcomes in patients through drug-drug interactions (DDIs). Absorption, distribution, metabolism, excretion and toxicity (ADMET) issues are the leading causes for the failure of a drug in the clinical trials. As details on their metabolism are known for just half of the approved drugs, a tool for reliable prediction of CYPs specificity is needed. The SuperCYPsPred web server is currently focused on five major CYPs isoenzymes, which includes CYP1A2, CYP2C19, CYP2D6, CYP2C9 and CYP3A4 that are responsible for more than 80% of the metabolism of clinical drugs. The prediction models for classification of the CYPs inhibition are based on well-established machine learning methods. The models were validated both on cross-validation and external validation sets and achieved good performance. The web server takes a 2D chemical structure as input and reports the CYP inhibition profile of the chemical for 10 models using different molecular fingerprints, along with confidence scores, similar compounds, known CYPs information of drugs-published in literature, detailed interaction profile of individual cytochromes including a DDIs table and an overall CYPs prediction radar chart (http://insilico-cyp.charite.de/SuperCYPsPred/). The web server does not require log in or registration and is free to use.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Software , Antidepressivos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Internet , Isoenzimas/química , Isoenzimas/metabolismo , Sertralina/farmacologia
17.
Cancer Chemother Pharmacol ; 85(4): 805-816, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32185484

RESUMO

PURPOSE: Metarrestin is a first-in-class pyrrolo-pyrimidine-derived small molecule targeting a marker of genome organization associated with metastasis and is currently in preclinical development as an anti-cancer agent. Here, we report the in vitro ADME characteristics and in vivo pharmacokinetic behavior of metarrestin. METHODS: Solubility, permeability, and efflux ratio as well as in vitro metabolism of metarrestin in hepatocytes, liver microsomes and S9 fractions, recombinant cytochrome P450 (CYP) enzymes, and potential for CYP inhibition were evaluated. Single dose pharmacokinetic profiles after intravenous and oral administration in mice, rat, dog, monkey, and mini-pig were obtained. Simple allometric scaling was applied to predict human pharmacokinetics. RESULTS: Metarrestin had an aqueous solubility of 150 µM at pH 7.4, high permeability in PAMPA and moderate efflux ratio in Caco-2 assays. The compound was metabolically stable in liver microsomes, S9 fractions, and hepatocytes from six species, including human. Metarrestin is a CYP3A4 substrate and, in mini-pigs, is also directly glucuronidated. Metarrestin did not show cytochrome P450 inhibitory activity. Plasma concentration-time profiles showed low to moderate clearance, ranging from 0.6 mL/min/kg in monkeys to 48 mL/min/kg in mice and moderate to high volume of distribution, ranging from 1.5 L/kg in monkeys to 17 L/kg in mice. Metarrestin has greater than 80% oral bioavailability in all species tested. The excretion of unchanged parent drug in urine was < 5% in dogs and < 1% in monkeys over collection periods of ≥ 144 h; in bile-duct cannulated rats, the excretion of unchanged drug was < 1% in urine and < 2% in bile over a collection period of 48 h. CONCLUSIONS: Metarrestin is a low clearance compound which has good bioavailability and large biodistribution after oral administration. Biotransformation appears to be the major elimination process for the parent drug. In vitro data suggest a low drug-drug interaction potential on CYP-mediated metabolism. Overall favorable ADME and PK properties support metarrestin's progression to clinical investigation.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Microssomos Hepáticos/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Pirróis/administração & dosagem , Pirróis/farmacocinética , Administração Oral , Animais , Biotransformação , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Feminino , Haplorrinos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura , Distribuição Tecidual
18.
Int J Mol Sci ; 21(4)2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32074957

RESUMO

Doxorubicin (DOX) is an effective chemotherapeutic agent used to treat a wide variety of malignancies. In addition to its multi-organ toxicity, DOX treatment has been shown to induce systemic inflammation in patients and experimental animals. Inflammation alters the expression of hepatic cytochrome P450 (CYP) enzymes, which play important roles in drug metabolism and DOX-induced toxicity. Significant sex differences have been reported in DOX-induced toxicity; however, sex differences in DOX-induced systemic inflammation and the potential effects on hepatic CYP expression have not been determined. In the current work, male and female C57Bl/6 mice were administered DOX (20 mg/kg by intraperitoneal injection), and groups of mice were sacrificed 24 and 72 h after DOX administration. DOX elicited a systemic inflammatory response in both male and female mice, but the inflammatory response was stronger in male mice. DOX altered the expression of hepatic CYP isoforms in a sex-dependent manner. Most notably, inhibition of Cyp2c29 and Cyp2e1 was stronger in male than in female mice, which paralleled the sex differences in systemic inflammation. Therefore, sex differences in DOX-induced systemic inflammation may lead to sexually dimorphic drug interactions, in addition to contributing to the previously reported sexual dimorphism in specific DOX-induced organ toxicity.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Doxorrubicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/enzimologia , Animais , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Feminino , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Injeções Intraperitoneais , Interleucina-6/sangue , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Caracteres Sexuais , Fator de Necrose Tumoral alfa/sangue
19.
PLoS One ; 15(2): e0229025, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32069299

RESUMO

Mutations in CYP4F22 cause autosomal recessive congenital ichthyosis (ARCI). However, less than 10% of all ARCI patients carry a mutation in CYP4F22. In order to identify the molecular basis of ARCI among our patients (a cohort of ninety-two Spanish individuals) we performed a mutational analysis using direct Sanger sequencing in combination with a multigene targeted NGS panel. From these, eight ARCI families (three of them with Moroccan origin) were found to carry five different CYP4F22 mutations, of which two were novel. Computational analysis showed that the mutations found were present in highly conserved residues of the protein and may affect its structure and function. Seven of the eight families were carriers of a highly recurrent CYP4F22 variant, c.1303C>T; p.(His435Tyr). A 12Mb haplotype was reconstructed in all c.1303C>T carriers by genotyping ten microsatellite markers flanking the CYP4F22 gene. A prevalent 2.52Mb haplotype was observed among Spanish carrier patients suggesting a recent common ancestor. A smaller core haplotype of 1.2Mb was shared by Spanish and Moroccan families. Different approaches were applied to estimate the time to the most recent common ancestor (TMRCA) of carrier patients with Spanish origin. The age of the mutation was calculated by using DMLE and BDMC2. The algorithms estimated that the c.1303C>T variant arose approximately 2925 to 4925 years ago, while Spanish carrier families derived from a common ancestor who lived in the XIII century. The present study reports five CYP4F22 mutations, two of them novel, increasing the number of CYP4F22 mutations currently listed. Additionally, our results suggest that the recurrent c.1303C>T change has a founder effect in Spanish population and c.1303C>T carrier families originated from a single ancestor with probable African ancestry.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Efeito Fundador , Genes Recessivos , Ictiose Lamelar/genética , Mutação , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Sistema Enzimático do Citocromo P-450/química , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Fenótipo , Conformação Proteica , Espanha , Relação Estrutura-Atividade
20.
Anal Bioanal Chem ; 412(8): 1729-1740, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32030490

RESUMO

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Espectrometria de Massas/métodos , Microssomos Hepáticos/enzimologia , Adulto , Idoso , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Feminino , Glucuronosiltransferase/química , Humanos , Masculino , Pessoa de Meia-Idade , Mapeamento de Peptídeos , Adulto Jovem
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