RESUMO
Acanthamoeba castellanii is a free-living amoeba that can cause severe eye and brain infections in humans. At present, there is no uniformly effective treatment for any of these infections. However, sterol 14α-demethylases (CYP51s), heme-containing cytochrome P450 enzymes, are known to be validated drug targets in pathogenic fungi and protozoa. The catalytically active P450 form of CYP51 from A. castellanii (AcCYP51) is stabilized against conversion to the inactive P420 form by dimerization. In contrast, Naegleria fowleri CYP51 (NfCYP51) is monomeric in its active P450 and inactive P420 forms. For these two CYP51 enzymes, we have investigated the interplay between the enzyme activity and oligomerization state using steady-state and time-resolved UV-visible absorption spectroscopy. In both enzymes, the P450 â P420 transition is favored under reducing conditions. The transition is accelerated at higher pH, which excludes a protonated thiol as the proximal ligand in P420. Displacement of the proximal thiolate ligand is also promoted by adding exogenous nitrogenous ligands (N-ligands) such as imidazole, isavuconazole, and clotrimazole that bind at the opposite, distal heme side. In AcCYP51, the P450 â P420 transition is faster in the monomer than in the dimer, indicating that the dimeric assembly is critical for stabilizing thiolate coordination to the heme and thus for sustaining AcCYP51 activity. The spectroscopic experiments were complemented with size-exclusion chromatography and X-ray crystallography studies. Collectively, our results indicate that effective inactivation of the AcCYP51 function by azole drugs is due to synergistic interference with AcCYP51 dimerization and promoting irreversible displacement of the proximal heme-thiolate ligand.
Assuntos
Acanthamoeba castellanii , Heme , Acanthamoeba castellanii/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Dimerização , Heme/química , Humanos , Ligantes , Nitrogênio/metabolismoRESUMO
The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 µM and 9.0 µM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 µM to 3.3 µM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.
Assuntos
Antivirais , Proteases 3C de Coronavírus , Proteases Semelhantes à Papaína de Coronavírus , Naftoquinonas , Inibidores de Proteases , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Antivirais/química , COVID-19 , Simulação de Acoplamento Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Papaína , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidoresRESUMO
Cytochromes P450 (P450, CYP) metabolize a wide variety of endogenous and exogenous lipophilic molecules, including most drugs. Sterol 14α-demethylase (CYP51) is a target for antifungal drugs known as conazoles. Using X-ray crystallography, we have discovered a domain-swap homodimerization mode in CYP51 from a human pathogen, Acanthamoeba castellanii CYP51 (AcCYP51). Recombinant AcCYP51 with a truncated transmembrane helix was purified as a heterogeneous mixture corresponding to the dimer and monomer units. Spectral analyses of these two populations have shown that the CO-bound ferrous form of the dimeric protein absorbed at 448 nm (catalytically competent form), whereas the monomeric form absorbed at 420 nm (catalytically incompetent form). AcCYP51 dimerized head-to-head via N-termini swapping, resulting in formation of a nonplanar protein-protein interface exceeding 2000 Å2 with a total solvation energy gain of -35.4 kcal/mol. In the dimer, the protomers faced each other through the F and G α-helices, thus blocking the substrate access channel. In the presence of the drugs clotrimazole and isavuconazole, the AcCYP51 drug complexes crystallized as monomers. Although clotrimazole-bound AcCYP51 adopted a typical CYP monomer structure, isavuconazole-bound AcCYP51 failed to refold 74 N-terminal residues. The failure of AcCYP51 to fully refold upon inhibitor binding in vivo would cause an irreversible loss of a structurally aberrant enzyme through proteolytic degradation. This assumption explains the superior potency of isavuconazole against A. castellanii The dimerization mode observed in this work is compatible with membrane association and may be relevant to other members of the CYP family of biologic, medical, and pharmacological importance. SIGNIFICANCE STATEMENT: We investigated the mechanism of action of antifungal drugs in the human pathogen Acanthamoeba castellanii. We discovered that the enzyme target [Acanthamoeba castellanii sterol 14α-demethylase (AcCYP51)] formed a dimer via an N-termini swap, whereas drug-bound AcCYP51 was monomeric. In the AcCYP51-isavuconazole complex, the protein target failed to refold 74 N-terminal residues, suggesting a fundamentally different mechanism of AcCYP51 inactivation than only blocking the active site. Proteolytic degradation of a structurally aberrant enzyme would explain the superior potency of isavuconazole against A. castellanii.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Amebíase/tratamento farmacológico , Proteínas de Protozoários/antagonistas & inibidores , Esterol 14-Desmetilase/metabolismo , Inibidores de 14-alfa Desmetilase/uso terapêutico , Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Ligação Proteica , Domínios Proteicos/fisiologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Proteólise/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestrutura , Piridinas/farmacologia , Piridinas/uso terapêutico , Proteínas Recombinantes , Esterol 14-Desmetilase/ultraestrutura , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
Cytochromes P450 (P450s) are nature's catalysts of choice for performing demanding and physiologically vital oxidation reactions. Biochemical characterization of these enzymes over the past decades has provided detailed mechanistic insight and highlighted the diversity of substrates P450s accommodate and the spectrum of oxidative transformations they catalyze. Previously, we discovered that the bacterial P450 MycCI from the mycinamicin biosynthetic pathway in Micromonospora griseorubida possesses an unusually broad substrate scope, whereas the homologous P450 from tylosin-producing Streptomyces fradiae (TylHI) exhibits a high degree of specificity for its native substrate. Here, using biochemical, structural, and computational approaches, we aimed to understand the molecular basis for the disparate reactivity profiles of these two P450s. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI strictly prefers 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. To help rationalize these results, we solved the X-ray crystal structure of TylHI in complex with its native substrate at 1.99-Å resolution and assayed several site-directed mutants. We also conducted molecular dynamics simulations of TylHI and MycCI and biochemically characterized a third P450 homolog from the chalcomycin biosynthetic pathway in Streptomyces bikiniensis These studies provided a basis for constructing P450 chimeras to gain further insight into the features dictating the differences in reaction profile among these structurally and functionally related enzymes, ultimately unveiling the central roles of key loop regions in influencing substrate binding and turnover. Our work highlights the complex nature of P450/substrate interactions and raises interesting questions regarding the evolution of functional diversity among biosynthetic enzymes.
Assuntos
Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Tilosina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Antifúngicos/farmacologia , Nitrilas/farmacologia , Piridinas/farmacologia , Triazóis/farmacologia , Inibidores de 14-alfa Desmetilase/farmacologia , Inibidores de 14-alfa Desmetilase/uso terapêutico , Acanthamoeba castellanii/crescimento & desenvolvimento , Amebíase/tratamento farmacológico , Amebíase/parasitologia , Amebicidas/uso terapêutico , Animais , Antifúngicos/uso terapêutico , Reposicionamento de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Nitrilas/uso terapêutico , Piridinas/uso terapêutico , Triazóis/uso terapêutico , Trofozoítos/efeitos dos fármacosRESUMO
Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8-Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenolâergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a "switch" between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8-Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity.
Assuntos
Naegleria fowleri/metabolismo , Esteróis/biossíntese , Sequência de Aminoácidos , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Barreira Hematoencefálica , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Colesterol/biossíntese , Descoberta de Drogas , Reposicionamento de Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/patogenicidade , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Esteroide Isomerases/antagonistas & inibidores , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismoRESUMO
The electron configuration of flavin cofactors, FMN and FAD, is a critical factor governing the reactivity of NADPH-cytochrome P450 reductase (CPR). The current view of electron transfer by the mammalian CPR, based on equilibrium redox potentials of the flavin cofactors, is that the two electron-reduced FMN hydroquinone (FMNH2), rather than one electron-reduced FMN semiquinone, serves as electron donor to the terminal protein acceptors. However, kinetic and thermodynamic studies on the CPR species originated from different organisms have shown that redox potentials measured at distinct electron transfer steps differ from redox potentials determined by equilibrium titration. Collectively, previous observations suggest that the short-lived transient semiquinone species may carry electrons in diflavin reductases. In this work, we have investigated spectroscopic properties of the CPR-bound FAD and FMN reduced at 77â¯K by radiolytically-generated thermalized electrons. Using UV-vis spectroscopy, we demonstrated that upon cryo-reduction of oxidized yeast CPR (yCPR) containing an equimolar ratio of both FAD and FMN, or FAD alone, neutral semiquinones were trapped at 77â¯K. During annealing at the elevated temperatures, unstable short-lived neutral semiquinones relaxed to spectroscopically distinct air-stable neutral semiquinones. This transition was independent of pH within the 6.0-10.7 range. Our data on yeast CPR are in line with the previous observations of others that the flavin short-lived transient semiquinone intermediates may have a role in the electron transfer by CPR at physiological conditions.
Assuntos
Flavina-Adenina Dinucleotídeo/análogos & derivados , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Temperatura , Leveduras/enzimologia , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Glucose Oxidase/metabolismo , Cinética , OxirreduçãoRESUMO
MycG is a P450 monooxygenase that catalyzes the sequential hydroxylation and epoxidation of mycinamicin IV (M-IV), the last two steps in the biosynthesis of mycinamicin II, a macrolide antibiotic isolated from Micromonospora griseorubida. The crystal structure of MycG with M-IV bound was previously determined but showed the bound substrate in an orientation that did not rationalize the observed regiochemistry of M-IV hydroxylation. Nuclear magnetic resonance paramagnetic relaxation enhancements provided evidence of an orientation of M-IV in the MycG active site more compatible with the observed chemistry, but substrate-induced changes in the enzyme structure were not characterized. We now describe the use of amide 1H-15N residual dipolar couplings as experimental restraints in solvated "soft annealing" molecular dynamics simulations to generate solution structural ensembles of M-IV-bound MycG. Chemical shift perturbations, hydrogen-deuterium exchange, and 15N relaxation behavior provide insight into the dynamic and electronic perturbations in the MycG structure in response to M-IV binding. The solution and crystallographic structures are compared, and the possibility that the crystallographic orientation of bound M-IV represents an inhibitory mode is discussed.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Simulação de Dinâmica Molecular , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Soluções , Especificidade por SubstratoRESUMO
The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.
Assuntos
Antiprotozoários/química , Auranofina/química , Entamoeba histolytica/química , Proteínas de Protozoários/química , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/química , Sequência de Aminoácidos , Antirreumáticos/química , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Dissulfetos/química , Entamoeba histolytica/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Mutação , Oxirredução , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Domínio Catalítico , Macrolídeos/metabolismo , Modelos Moleculares , Oxirredução , Ligação ProteicaRESUMO
Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Amino Açúcares/química , Amino Açúcares/metabolismo , Biocatálise , Sistema Enzimático do Citocromo P-450/química , Hidroxilação , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Moleculares , Estereoisomerismo , Especificidade por SubstratoRESUMO
Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4-aminopyridyl-based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub-nanomolar compound 3, have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X-ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground-state conformation of CYP51 drug-target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Esterol 14-Desmetilase/metabolismo , Sulfonamidas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Inibidores de 14-alfa Desmetilase/síntese química , Inibidores de 14-alfa Desmetilase/química , Animais , Relação Dose-Resposta a Droga , Camundongos , Modelos Moleculares , Estrutura Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma cruzi/enzimologiaRESUMO
The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ~4 Å of the oxygen of iron-ligated water.
Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Macrolídeos/metabolismo , Micromonospora/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Compostos de Epóxi/metabolismo , Glicosilação , Hidroxilação , Macrolídeos/química , Espectroscopia de Ressonância Magnética , Metilação , Micromonospora/genética , Micromonospora/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Oxirredução , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Degradation of the cholesterol side-chain in Mycobacterium tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis mc(2) 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures of both substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatisâ Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.
Assuntos
Colesterol/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Antifúngicos/metabolismo , Azóis/metabolismo , Sítios de Ligação , Colestenonas/metabolismo , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Hidroxilação , Modelos Moleculares , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Oxirredução , Estrutura Terciária de ProteínaRESUMO
Inhibition of the Trypanosoma cruzi cysteine protease cruzain has been proposed as a therapeutic approach for the treatment of Chagas' disease. Among the best-studied cruzain inhibitors to date is the vinylsulfone K777 (1), which has proven effective in animal models of Chagas' disease. Recent structure-activity studies aimed at addressing potential liabilities of 1 have now produced analogues such as N-[(2S)-1-[[(E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-3-(4-methylphenyl)-1-oxopropan-2-yl]pyridine-4-carboxamide (4), which is trypanocidal at ten-fold lower concentrations than for 1. We now find that the trypanocidal activity of 4 derives primarily from the inhibition of T. cruzi 14-α-demethylase (TcCYP51), a cytochrome P450 enzyme involved in the biosynthesis of ergosterol in the parasite. Compound 4 also inhibits mammalian CYP isoforms but is trypanocidal at concentrations below those required to significantly inhibit mammalian CYPs in vitro. A chemical-proteomics approach employing an activity-based probe derived from 1 was used to identify mammalian cathepsin B as a potentially important off-target of 1 and 4. Computational docking studies and the evaluation of truncated analogues of 4 reveal structural determinants for TcCYP51 binding, information that will be useful in further optimization of this new class of inhibitors.
RESUMO
Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC50 ≤ 10 µM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC50 and KD compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.
Assuntos
Miconazol , Naegleria fowleri , Inibidores de 14-alfa Desmetilase/farmacologia , Descoberta de DrogasRESUMO
Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 µM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.
Assuntos
COVID-19 , Tiossemicarbazonas , Humanos , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , Tiossemicarbazonas/farmacologia , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteínas não Estruturais ViraisRESUMO
Diverse oxygenation patterns of natural products generated by secondary metabolic pathways in microorganisms and plants are largely achieved through the tailoring reactions catalysed by cytochrome P450 enzymes (P450s). P450s are a large family of oxidative hemoproteins found in all life forms from prokaryotes to humans. Understanding the reactivity and selectivity of these fascinating C-H bond-activating catalysts will advance their use in generating valuable pharmaceuticals and products for medicine, agriculture and industry. A major strength of this P450 group is its set of established enzyme-substrate relationships, the source of the most detailed knowledge on how P450 enzymes work. Engineering microbial-derived P450 enzymes to accommodate alternative substrates and add new functions continues to be an important near- and long-term practical goal driving the structural characterization of these molecules. Understanding the natural evolution of P450 structure-function should accelerate metabolic engineering and directed evolutionary approaches to enhance diversification of natural product structures and other biosynthetic applications.
Assuntos
Produtos Biológicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Produtos Biológicos/química , Catálise , Sistema Enzimático do Citocromo P-450/química , Fungos/enzimologia , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Plantas/enzimologiaRESUMO
Mycobacterium tuberculosis (Mtb) produces a variety of methyl-branched lipids that serve important functions, including modulating the immune response during pathogenesis and contributing to a robust cell wall that is impermeable to many chemical agents. Here, we report characterization of Mtb CYP124 (Rv2266) that includes demonstration of preferential oxidation of methyl-branched lipids. Spectrophotometric titrations and analysis of reaction products indicate that CYP124 tightly binds and hydroxylates these substrates at the chemically disfavored omega-position. We also report X-ray crystal structures of the ligand-free and phytanic acid-bound protein at a resolution of 1.5 A and 2.1 A, respectively, which provide structural insights into a cytochrome P450 with predominant omega-hydroxylase activity. The structures of ligand-free and substrate-bound CYP124 reveal several differences induced by substrate binding, including reorganization of the I helix and closure of the active site by elements of the F, G, and D helices that bind the substrate and exclude solvent from the hydrophobic active site cavity. The observed regiospecific catalytic activity suggests roles of CYP124 in the physiological oxidation of relevant Mtb methyl-branched lipids. The enzymatic specificity and structures reported here provide a scaffold for the design and testing of specific inhibitors of CYP124.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocromo P-450 CYP4A/química , Citocromo P-450 CYP4A/metabolismo , Mycobacterium tuberculosis/enzimologia , Biocatálise , Domínio Catalítico , Hidroxilação , Ligantes , Modelos Moleculares , Ácido Fitânico/química , Ácido Fitânico/metabolismo , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique desosamine-anchoring functionality via a heretofore under-explored "substrate engineering" strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the desosamine glycoside via an acetal linkage (referred to as "carbolides") in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.