RESUMO
Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high levels of glucose. Flux through GK is also responsible for reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can activate GK could provide a therapeutic benefit. Herein we report the further structure activity studies of a novel series of glucokinase activators (GKA). These studies led to the identification of pyridine 72 as a potent GKA that lowered post-prandial glucose in normal C57BL/6J mice, and after 14d dosing in ob/ob mice.
Assuntos
Ativadores de Enzimas/química , Glucoquinase/química , Hipoglicemiantes/química , Animais , Sítios de Ligação , Glicemia/análise , Cristalografia por Raios X , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/uso terapêutico , Glucoquinase/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/metabolismo , Hipoglicemiantes/uso terapêutico , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Tiadiazóis/química , Tiadiazóis/metabolismoRESUMO
P450 family 4 fatty acid ω-hydroxylases preferentially oxygenate primary C-H bonds over adjacent, energetically favored secondary C-H bonds, but the mechanism explaining this intriguing preference is unclear. To this end, the structure of rabbit P450 4B1 complexed with its substrate octane was determined by X-ray crystallography to define features of the active site that contribute to a preference for ω-hydroxylation. The structure indicated that octane is bound in a narrow active-site cavity that limits access of the secondary C-H bond to the reactive intermediate. A highly conserved sequence motif on helix I contributes to positioning the terminal carbon of octane for ω-hydroxylation. Glu-310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu-310 contributes to substrate positioning. The preference for ω-hydroxylation was decreased in an E310A mutant having a shorter side chain, but the overall rates of metabolism were retained. E310D and E310Q substitutions having longer side chains exhibit lower overall rates, likely due to higher conformational entropy for these residues, but they retained high preferences for octane ω-hydroxylation. Sequence comparisons indicated that active-site residues constraining octane for ω-hydroxylation are conserved in family 4 P450s. Moreover, the heme 7-propionate is positioned in the active site and provides additional restraints on substrate binding. In conclusion, P450 4B1 exhibits structural adaptations for ω-hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I motif that is associated with selective oxygenation of unactivated primary C-H bonds.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Sequência Conservada , Octanos/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Heme/química , Heme/metabolismo , Hidroxilação , Conformação Proteica , Coelhos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Mutant BRAFV600E is one of the most common oncogenic drivers in metastatic melanoma. While first generation BRAFV600E inhibitors are capable of controlling tumors systemically, they are unable to adequately treat tumors that have metastasized to the brain due to insufficient penetration across the blood-brain barrier (BBB). Through a combination of structure-based drug design (SBDD) and the optimization of physiochemical properties to enhance BBB penetration, we herein report the discovery of the brain-penetrant BRAFV600E inhibitor PF-07284890 (ARRY-461). In mice studies, ARRY-461 proved to be highly brain-penetrant and was able to drive regressions of A375 BRAFV600E tumors implanted both subcutaneously and intracranially. Based on compelling preclinical safety and efficacy studies, ARRY-461 was progressed into a Phase 1 A/B clinical trial (NCT04543188).
Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Humanos , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Relação Estrutura-Atividade , Ratos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , MasculinoRESUMO
RAF inhibitors have transformed treatment for BRAF V600-mutant cancer patients, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAF V600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAF V600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in BRAF-mutant cancer patients refractory to approved RAF inhibitors.
RESUMO
Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE: The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.See related commentary by Klempner and Hata, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Acetonitrilas/uso terapêutico , Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirrolidinas/uso terapêutico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Apoptose , Proliferação de Células , Ensaios Clínicos Fase I como Assunto , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , Pirimidinas , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Genfibrozila/análogos & derivados , Glucuronatos/química , Heme/química , Alquilação , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Simulação por Computador , Citocromo P-450 CYP2C8 , Genfibrozila/química , Genfibrozila/metabolismo , Genfibrozila/farmacologia , Genfibrozila/toxicidade , Glucuronatos/farmacologia , Glucuronatos/toxicidade , Humanos , Hipolipemiantes/metabolismo , Espectrometria de Massas , Oxirredução , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
KRAS is the most frequently mutated driver oncogene in human cancer, and KRAS mutations are commonly associated with poor prognosis and resistance to standard treatment. The ability to effectively target and block the function of mutated KRAS has remained elusive despite decades of research. Recent findings have demonstrated that directly targeting KRAS-G12C with electrophilic small molecules that covalently modify the mutated codon 12 cysteine is feasible. We have discovered a series of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRAS-G12C with in vivo activity. The PK/PD and efficacy of compound 13 will be highlighted.
RESUMO
Glucokinase (GK) is the rate-limiting step for insulin release from the pancreas in response to high levels of glucose. Flux through GK also contributes to reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can allosterically activate GK may address this issue. Herein we report the identification and initial optimization of a novel series of glucokinase activators (GKAs). Optimization led to the identification of 33 as a compound that displayed activity in an oral glucose tolerance test (OGTT) in normal and diabetic mice.
Assuntos
Ativadores de Enzimas/síntese química , Glucoquinase/metabolismo , Piridinas/síntese química , Ureia/análogos & derivados , Animais , Descoberta de Drogas , Ativadores de Enzimas/farmacologia , Teste de Tolerância a Glucose , Camundongos Endogâmicos C57BL , Piridinas/farmacologiaRESUMO
Glucokinase (GK) is a hexokinase isozyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate. Glucokinase activators are being investigated as potential diabetes therapies because of their effects on hepatic glucose output and/or insulin secretion. Here, we have examined the efficacy and mechanisms of action of a novel glucokinase activator, GKA23. In vitro, GKA23 increased the affinity of rat and mouse glucokinase for glucose, and increased glucose uptake in primary rat hepatocytes. In vivo, GKA23 treatment improved glucose homeostasis in rats by enhancing beta cell insulin secretion and suppressing hepatic glucose production. Sub-chronic GKA23 treatment of mice fed a high-fat diet resulted in improved glucose homeostasis and lipid profile.
Assuntos
Aminopiridinas/química , Ativadores de Enzimas/química , Glucoquinase/metabolismo , Tiadiazóis/química , Animais , Área Sob a Curva , Glicemia/metabolismo , Catálise , Diabetes Mellitus Experimental/tratamento farmacológico , Glucose/metabolismo , Teste de Tolerância a Glucose , Hepatócitos/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Cinética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Glucokinase (GK) activators represent a class of type 2 diabetes therapeutics actively pursued due to the central role that GK plays in regulating glucose homeostasis. Herein we report a novel C5-alkyl-2-methylurea-substituted pyridine series of GK activators derived from our previously reported thiazolylamino pyridine series. Our efforts in optimizing potency, enzyme kinetic properties, and metabolic stability led to the identification of compound 26 (AM-9514). This analogue showed a favorable combination of in vitro potency, enzyme kinetic properties, acceptable pharmacokinetic profiles in preclinical species, and robust efficacy in a rodent PD model.
RESUMO
Cytochrome P450s in the CYP4 family covalently bind their heme prosthetic group to a conserved acidic I-helix residue via an autocatalytic oxidation. This study was designed to evaluate the source of oxygen atoms in the covalent ester link in CYP4B1 enzymes labeled with [18O]glutamate and [18O]aspartate. The fate of the heavy isotope was then traced into wild-type CYP4B1 or the E310D mutant-derived 5-hydroxyhemes. Glutamate-containing tryptic peptides of wild-type CYP4B1 were found labeled to a level of 11-13% 18O. Base hydrolysis of labeled protein released 5-hydroxyheme which contained 12.8 +/- 1.9% 18O. Aspartate-containing peptides of the E310D mutant were labeled with 6.0-6.5% 18O, but as expected, no label was transmitted to recovered 5-hydroxyheme. These data demonstrate that the oxygen atom in 5-hydroxyheme derived from wild-type CYP4B1 originates in Glu310. Stoichiometric incorporation of the heavy isotope from the wild-type enzyme supports a perferryl-initiated carbocation mechanism for covalent heme formation in CYP4B1.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Heme/química , Sequência de Aminoácidos , Hidrocarboneto de Aril Hidroxilases/genética , Ácido Aspártico , Sítios de Ligação , Ésteres , Ácido Glutâmico , Cinética , Espectrometria de Massas , Fragmentos de Peptídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , TripsinaRESUMO
Time-dependent inactivation of cytochrome P450s is typically a result of substrate bioactivation to form reactive species that subsequently alkylate the heme group, apoprotein, or both. The chemical identity of many reactive intermediates is generally proposed based on the products of trapping reactions with nucleophilic agents as only a few P450-drug adducts have been directly characterized. We describe the use of mass spectrometry to show that a single equivalent of raloxifene is bound to the intact P450 apoprotein. Furthermore, mass analysis of peptides following digestion with proteinase K revealed that the covalently bound drug is localized to residue Cys239. A mass shift of 471 Da to the intact protein and peptide, relative to control samples, indicated that time-dependent inactivation of P450 3A4 occurred through the raloxifene diquinone methide intermediately prior to nucleophilic attack of the sulfur of Cys239. Association between raloxifene adduction to P450 3A4 apoprotein and the observed time-dependent inactivation was further investigated with the use of cysteine-specific modifying reagents. When P450 3A4 was treated with iodoacetamide or N-(1-pyrene)iodoacetamide, which alkylated residue Cys239 exclusively, time-dependent inactivation of P450 3A4 by raloxifene was prevented. The change in protein mass of 471 Da combined with the protection from inactivation that occurred through pre-alkylation of Cys239 provided conclusive evidence that raloxifene-mediated P450 3A4 inactivation occurred through the bioactivation of raloxifene to the diquinone methide and subsequent alkylation of Cys239.
Assuntos
Apoproteínas/metabolismo , Cisteína/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Cloridrato de Raloxifeno/farmacologia , Alquilantes/química , Alquilantes/farmacologia , Alquilação/efeitos dos fármacos , Apoproteínas/química , Sítios de Ligação , Cisteína/química , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Endopeptidase K/química , Endopeptidase K/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Glutationa/química , Glutationa/metabolismo , Iodoacetamida/análogos & derivados , Iodoacetamida/química , Iodoacetamida/farmacologia , Cinética , Midazolam/química , Midazolam/farmacologia , Estrutura Molecular , Quinolinas/química , Quinolinas/metabolismo , Cloridrato de Raloxifeno/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
CYP4B1 belongs to the mammalian CYP4 enzyme family that also includes CYP4A, 4F, 4V, 4X, and 4Z subfamilies. CYP4B1 shares with other CYP4 proteins a capacity to omega-hydroxylate medium-chain fatty acids, which may be related to an endogenous role for the enzyme. CYP4B1 also participates in the metabolism of certain xenobiotics that are protoxic, including valproic acid, 3-methylindole, 4-ipomeanol, 3-methoxy-4-aminoazobenzene, and numerous aromatic amines. Although these compounds have little in common structurally or chemically, their metabolism by CYP4B1 leads to tissue-specific toxicities in several experimental animals. The bioactivation capabilities of rabbit CYP4B1 have also attracted attention in the cancer community and form the basis of a potential therapeutic strategy involving prodrug activation by the CYP4B1 transgene. The metabolic capabilities of human CYP4B1 are less clear due to difficulties in heterologous expression and existence of alternatively spliced products. Also, many CYP4B1 enzymes covalently bind their heme, a posttranslational modification unique to the CYP4 family of P450s, but common to the mammalian peroxidases. These varied characteristics render CYP4B1 an interesting and enigmatic investigational target.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Xenobióticos/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/química , Ativação Enzimática/efeitos dos fármacos , Heme/química , Heme/metabolismo , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Xenobióticos/farmacologiaRESUMO
4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromolecules. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liquid chromatography/electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping molecule. Reaction mixtures containing equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resolution mass spectrometry and two-dimensional NMR analysis following large-scale isolation of the biologically derived material provided evidence for an N-substituted cysteinyl pyrrole derivative of IPO, analogous to that characterized previously in model chemical studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P450/30 min. A panel of 14 commercially available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were observed with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s.
Assuntos
Aldeídos/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Terpenos/metabolismo , Acetilcisteína/química , Aldeídos/química , Animais , Antineoplásicos/farmacologia , Biotransformação , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/química , Glutationa/metabolismo , Humanos , Fígado/metabolismo , Pulmão/metabolismo , Lisina/química , NADP/química , Pirróis/química , Pirróis/metabolismo , Coelhos , Espectrometria de Massas por Ionização por Electrospray , Terpenos/toxicidadeRESUMO
Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality. Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Heme/metabolismo , Cromatografia Líquida de Alta Pressão , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Escherichia coli/genética , Heme/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/isolamento & purificação , Sequência de Bases , Benzoflavonas/metabolismo , Bioquímica/métodos , Membrana Celular/metabolismo , Estabilidade Enzimática , Expressão Gênica , Ácidos Láuricos/metabolismo , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Recently we found that CYP4B1, and several other members of the CYP4 family of enzymes, are covalently linked to their prosthetic heme group through an ester linkage. In the current study, we mutated a conserved CYP4 I-helix residue, E310 in rabbit CYP4B1, to glycine, alanine, and aspartate to examine the effect of these mutations on the extent of covalent heme binding and catalysis. All mutants expressed well in insect cells and were isolated as a mixture of monomeric and dimeric forms as determined by LC/ESI-MS of the intact proteins. Rates of metabolism decreased in the order E310 > A310 >> G310 > D310, with the A310 and G310 mutants exhibiting alterations in regioselectivity for omega-1 and omega-2 hydroxylation of lauric acid, respectively. In marked contrast to the wild-type E310 enzyme, the G310, A310, and D310 mutants did not bind heme covalently. Uniquely, the acid-dissociable heme obtained from the D310 mutant contained an additional 16 amu relative to heme and exhibited the same chromatographic behavior as the monohydroxyheme species released upon base treatment of the covalently linked wild-type enzyme. Expression studies with H(2)(18)O demonstrated incorporation of the heavy isotope from the media into the monohydroxyheme isolated from the D310 mutant at a molar ratio of approximately 0.8:1. These data show (i) that E310 serves as the site of covalent attachment of heme to the protein backbone of rabbit CYP4B1; (ii) this I-helix glutamate residue influences substrate orientation in the active site of CYP4B1; and (iii) the mechanism of covalent heme attachment most likely involves a carbocation species located on the porphyrin.