RESUMO
3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), a natural metabolite of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), exhibits potent vitamin D receptor (VDR)-mediated actions such as inhibition of keratinocyte growth or suppression of parathyroid hormone secretion. These VDR-mediated actions of 3-epi-1α,25(OH)2D3 needed an explanation as 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, exhibits low affinity towards VDR. Metabolic stability of 3-epi-1α,25(OH)2D3 over 1α,25(OH)2D3 has been hypothesized as a possible explanation. To provide further support for this hypothesis, we now performed comparative metabolism studies between 3-epi-1α,25(OH)2D3 and 1α,25(OH)2D3 using both the technique of isolated rat kidney perfusion and purified rat CYP24A1 in a cell-free reconstituted system. For the first time, these studies resulted in the isolation and identification of 3-epi-calcitroic acid as the final inactive metabolite of 3-epi-1α,25(OH)2D3 produced by rat CYP24A1. Furthermore, under identical experimental conditions, it was noted that the amount of 3-epi-calcitroic acid produced from 3-epi-1α,25(OH)2D3 is threefold less than that of calcitroic acid, the analogous final inactive metabolite produced from 1α,25(OH)2D3 . This key observation finally led us to conclude that the rate of overall side-chain oxidation of 3-epi-1α,25(OH)2D3 by rat CYP24A1 leading to its final inactivation is slower than that of 1α,25(OH)2D3. To elucidate the mechanism responsible for this important finding, we performed a molecular docking analysis using the crystal structure of rat CYP24A1. Docking results suggest that 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, binds to CYP24A1 in an alternate configuration that destabilizes the formation of the enzyme-substrate complex sufficiently to slow the rate at which 3-epi-1α,25(OH)2D3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid.
Assuntos
Hidroxicolecalciferóis/metabolismo , Simulação de Dinâmica Molecular , Esteroide Hidroxilases/metabolismo , Vitamina D/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Ratos , Vitamina D/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
BACKGROUND: The 1α,25-dihydroxy-3-epi-vitamin-D3 (1α,25(OH)2-3-epi-D3), a natural metabolite of the seco-steroid vitamin D3, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1α,25(OH)2-3-epi-D3 is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)2D3. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD). METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)2D3. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)2-3-epi-D3 in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)2D3. CONCLUSIONS/SIGNIFICANCE: The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3.
Assuntos
Colecalciferol/síntese química , Colecalciferol/farmacologia , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colecalciferol/análogos & derivados , Colecalciferol/química , Cristalografia por Raios X , Células HL-60 , Humanos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D3 (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D3 (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D3 (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D3 (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.
Assuntos
Esteroide Hidroxilases/metabolismo , Vitamina D/análogos & derivados , Animais , Cromatografia Gasosa-Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Ratos , Esteroide Hidroxilases/química , Vitamina D/química , Vitamina D/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
From earliest development on, the vitamin D receptor (VDR) is expressed in most cells of the mammalian body. The VDR is a nuclear, ligand-induced transcription factor that regulates in complex with hormonally active vitamin D the expression of more than 900 genes involved in a wide array of physiological functions (e.g. calcium homeostasis, growth control, differentiation, cognition, immune response, etc.). Accordingly, severe health problems are associated to vitamin deficiencies. Synthesis of the major active form 1α,25(OH)2D3 from vitamin D and subsequent metabolism are exclusively controlled by specific P450-forms. Synthesis, a two-step process, starts with a 25-hydroxylation primarily by CYP2R1 (CYP27A1, CYP2J2, and CYP3A4 may also contribute) and a subsequent 1α-hydroxylation via CYP27B1. Circulating in the bloodstream, 1α,25(OH)2D3 acts at sites of VDR expression (target sites) in an endocrine way. However, it is also capable of autocrine/paracrine functions since various target tissues are fully competent in 1α,25(OH)2D3 synthesis, as illustrated by three examples. 1α,25(OH)2D3 levels are short-lived: the hormone upregulates its rapid metabolism by CYP24A1 that attacks repeatedly the vitamin D C20â27 side chain, thereby producing a complex cascade of transient metabolites with increasing polarity. Most of these metabolites still retain 1α,25(OH)2D3-like activities on the VDR, contributing to the overall effect that is commonly attributed to 1α,25(OH)2D3. As selective inhibitors of CYP24A1 increase the lifetime and thereby the function of vitamin D metabolites, they will help exploring whether and which intrinsic activities distinct metabolites possess. It appears likely that this strategy may unmask important regulators of new functions.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Família 2 do Citocromo P450 , Humanos , Hidroxilação , Redes e Vias Metabólicas , Estrutura Molecular , Vitamina D/químicaRESUMO
The ubiquitously expressed natural polyamines putrescine, spermidine, and spermine are small, flexible cationic compounds that exert pleiotropic actions on various regulatory systems and, accordingly, are essentially involved in diverse life functions. These roles of polyamines result from their capability to interact with negatively charged regions of all major classes of biomolecules, which might act in response by changing their structures and functions. The present review deals with polyamine-protein interactions, thereby focusing on mammalian proteins. We discuss the various modes in which polyamines can interact with proteins, describe major types of affected functions illustrated by representative examples of involved proteins, and support information with respective structural evidence from elucidated three-dimensional structures. A specific focus is put on polyamine interactions at protein surfaces that can modulate the aggregation of proteins to organized structural networks as well as to toxic aggregates and, moreover, can play a role in important transient protein-protein interactions.
RESUMO
Modulations of protein-protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein-protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein-protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein-protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine>spermidine>putrescine) at the AdR-Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein-protein interactions.
Assuntos
Poliaminas/química , Poliaminas/farmacologia , Proteínas/química , Adrenodoxina/química , Adrenodoxina/genética , Adrenodoxina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Catálise/efeitos dos fármacos , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Oxirredução/efeitos dos fármacos , Poliaminas/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas/metabolismo , Putrescina/química , Putrescina/metabolismo , Putrescina/farmacologia , Espermidina/química , Espermidina/metabolismo , Espermidina/farmacologia , Espermina/química , Espermina/metabolismo , Espermina/farmacologia , Eletricidade EstáticaRESUMO
Sterol 14alpha-demethylases (CYP51) serve as primary targets for antifungal drugs, and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands that demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.
Assuntos
Antiprotozoários/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Oxirredutases/antagonistas & inibidores , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Tripanossomíase/tratamento farmacológico , Animais , Antiprotozoários/uso terapêutico , Esterol 14-Desmetilase , Especificidade por Substrato , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/citologia , Trypanosoma cruzi/enzimologiaRESUMO
Mammalian cytochromes P450 have been shown to play highly important roles in the metabolism of drugs and xenobiotics as well as in the biosynthesis of a variety of endogenous compounds, many of them displaying hormonal function. The role of P450s as therapeutic targets is still inadequately recognized although several P450 inhibitors became efficient drugs that even reached blockbuster status. Here, we try to give a comprehensive overview on cytochromes P450s, which are already well-established targets - particularly focussing on the treatment of infectious diseases, metabolic disorders and cancer - and on those, which have a high potential to become successful targets. In addition, the design of inhibitors of cytochromes P450 will be discussed.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Animais , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/fisiologia , Desenho de Fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologiaRESUMO
BACKGROUND: Levels of active vitamin D (VD) are controlled by synthesis via CYP27B1 and self-induced metabolism by CYP24A1. Unbalanced high CYP24A1 expression due to induction by diverse endogenous compounds and xenobiotics, and amplification found in various tumours, might lead to local VD deficiency, thereby causing/reinforcing disorders. MATERIALS AND METHODS: Using primary human keratinocytes, CYP24A1 expression was examined at the mRNA level by dot-blot and Northern blot hybridization, and at the enzyme activity level by analysing HPLC profiles from incubations with 3H-labelled VD metabolites. RESULTS: We have developed a one-step protocol to screen test compounds for potent inhibition of CYP24A1 along with selectivity over CYP27B1 and adequate metabolic stability. These inhibitors amplified hormone levels and, thereby, its function, indicated by increased CYP24A1 expression. Moreover, they stabilized the expression of a CYP24A1 splice variant, possibly serving as a buffer of VD metabolites. In addition, a low abundant, constitutive 24-hydroxylase, active in the low nanomolar range is described. CONCLUSION: Selective CYP24A1 inhibitors could herald a new era for vitamin D research, as well as for therapeutic application. Inhibitors may be used as single entities or in combination with low doses of potent analogs to prevent and treat various defects of growth and differentiation, and neuro-immuno-endocrine disorders.
Assuntos
Inibidores Enzimáticos/farmacologia , Esteroide Hidroxilases/antagonistas & inibidores , Vitamina D/antagonistas & inibidores , Vitamina D/metabolismo , Processamento Alternativo , Animais , Bovinos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Esteroide Hidroxilases/biossíntese , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.
Assuntos
Calcifediol/análogos & derivados , Calcifediol/metabolismo , Calcitriol/análogos & derivados , Calcitriol/metabolismo , Queratinócitos/metabolismo , Animais , Catálise , Linhagem Celular , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Meios de Cultura Livres de Soro , Humanos , Hidroxilação , Espectrometria de Massas , Ratos , Proteínas Recombinantes/metabolismoRESUMO
1alpha,25(OH)2D3 exerts antiproliferative, differentiating effects on many cell types, including cancer tissues. In most of its target cells, levels of 1alpha,25(OH)2D3 are regulated by local synthesis via CYP27B and metabolism via CYP24. Rapidly induced by vitamin D, CYP24 repeatedly hydroxylates the vitamin D side chain and ultimately terminates hormonal activity. Aiming at increased hormone levels, lifetime and function, numerous vitamin D analogs have been synthesized with structural modifications, which impede oxidation of the vitamin D side chain. Our group followed a different strategy, namely, blocking 1,25(OH)2D3 metabolism with inhibitors of CYP24. As appropriate inhibitors, we exploited compounds termed azoles, which directly bind to the heme iron of the CYPs via an azole nitrogen and to other parts of the substrate site. We synthesized some 400 azoles and tested their potential to selectively inhibit CYP24, but not hormone synthesis by the related CYP27B. Using primary human keratinocyte cultures as the source of CYP24 and CYP27, we discovered some 50 inhibitors of CYP24 with IC50 values in the nanomole range and selectivities up to 60-fold. As the first representative of selective CYP24 inhibitors, VID400 underwent preclinical development. In human keratinocytes, VID400 stabilized levels of endogenously produced 1alpha,25(OH)2D3, and thereby strongly amplified and prolonged expression of CYP24, a surrogate marker of hormonal function. In parallel, antiproliferative activity showed up at 100-fold or more lower concentrations of 1alpha,25(OH)2D3. This data suggests that CYP24 inhibitors could become attractive drugs in antiproliferative therapy, used as single entities to increase or extend endogenous hormone function or in combination with low doses of potent analogs. Moreover, we used selective inhibitors as valuable tools to (a) elucidate regulatory mechanisms of vitamin D synthesis and metabolism, (b) determine intrinsic activities of the otherwise highly transient vitamin D metabolites and (c) model the active sites of CYP24 and CYP27B.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Queratinócitos/efeitos dos fármacos , Esteroide Hidroxilases/antagonistas & inibidores , Vitamina D/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Queratinócitos/enzimologia , Esteroide Hidroxilases/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
Gliomas are the most common malignant tumors in brain. Recent studies demonstrate the capacity of 1alpha,25(OH)2D3 to specifically induce cell death (apoptosis) in model glioma cell lines and in primary cultures from tumor tissue, but not in primary astrocytes. In spite of this promising activity, a broad therapeutic application of vitamin D metabolites and analogs is still restricted because of their poor bioavailability and their hypercalcemic actions. Compared to 1alpha,25(OH)2D3, its natural 3alpha-epimer exhibits far higher metabolic stability and a reduced calcemic effect. Focusing on a possible therapeutic advantage of the 3alpha-conformation, we have examined the apoptotic potential of a representative set of vitamin D analogs, each of them in the 3alpha- and 3beta-conformation, and of natural vitamin D metabolites in the rat C6 glioma cell line. Exposure of these cells to the synthetic analogs resulted in all cases in a pronounced reduction of cell density (tested by incorporation of neutral red) and induction of apoptosis, monitored by staining nuclei with Hoechst 33258 dye and by following DNA fragmentation by capillary electrophoresis. The 3alpha-epimers showed equivalent or even higher activity on C6 cells than their respective 3beta forms. For their potent effects on growth and apoptosis of tumor cells and their high metabolic stability combined with a low calcemic potential, we speculate that these 3a-epimers could provide advantages for a prospective treatment of glioma.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Calcitriol/farmacologia , Glioma/patologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Calcitriol/análogos & derivados , Glioma/tratamento farmacológico , Humanos , Ratos , Células Tumorais CultivadasRESUMO
Aiming at new drugs to efficiently treat diseases, in which either increased or decreased levels of active vitamin D are desirable, we have designed some 400 structurally different azole-type inhibitors and examined their capacity to selectively block vitamin D metabolism by CYP24 or synthesis by CYP27B, in human keratinocytes. Based on resulting data, we built pharmacophore models of the active sites using commercial software. The overlay of potent selective compounds indicated similar docking modes in the two-substrate pockets and allowed for identification of bioactive conformations. Superimposing these bioactive conformations with low energy conformers of 25(OH)D(3) suggested that the substrate-mimicked by strong inhibitors in size, shape and lipophilic character-binds to both enzymes in 6s-trans configuration. Pharmacophoric models implied a similar geometry of the substrate sites, nevertheless specific features of CYP24 and CYP27B could be defined. Bulky substituents in alpha-position to the azole caused selectivity for CYP24, whereas bulky substituents in beta-position could result in selectivity for CYP27B. Moreover, studies with small sterically restricted inhibitors revealed a probable location of the 3-OH-group of 25(OH)D(3) in CYP27B. In the absence of crystal structures, our inhibitors are valuable tools to model and understand the active sites of vitamin D hydroxylases, resulting in the design of powerful, selective therapeutics.