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1.
J Med Chem ; 62(20): 9008-9025, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31550156

RESUMO

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a "chromatin or histone code". Proteins that contain "reader" domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional coactivator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a role in cancer related inflammation and/or cancer metastasis.

2.
Nat Chem Biol ; 15(5): 519-528, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962627

RESUMO

Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.


Assuntos
Benzimidazóis/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Reprogramação Celular/efeitos dos fármacos , Proteína p300 Associada a E1A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Isoxazóis/farmacologia , Oxazepinas/farmacologia , Piperidinas/farmacologia , Benzimidazóis/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Inibidores Enzimáticos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isoxazóis/química , Estrutura Molecular , Oxazepinas/química , Piperidinas/química , Domínios Proteicos/efeitos dos fármacos
3.
J Steroid Biochem Mol Biol ; 189: 218-227, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30769091

RESUMO

Steroid hormones, including glucocorticoids and androgens, have potent actions to regulate many cellular processes within the liver. The steroid A-ring reductase, 5ß-reductase (AKR1D1), is predominantly expressed in the liver, where it inactivates steroid hormones and, in addition, plays a crucial role in bile acid synthesis. However, the precise functional role of AKR1D1 to regulate steroid hormone action in vitro has not been demonstrated. We have therefore hypothesised that genetic manipulation of AKR1D1 has the potential to regulate glucocorticoid availability and action in human hepatocytes. In both liver (HepG2) and non-liver cell (HEK293) lines, AKR1D1 over-expression increased glucocorticoid clearance with a concomitant decrease in the activation of the glucocorticoid receptor and the down-stream expression of glucocorticoid target genes. Conversely, knockdown of AKR1D1 using siRNA decreased glucocorticoid clearance and reduced the generation of 5ß-reduced metabolites. In addition, the two 5α-reductase inhibitors finasteride and dutasteride failed to effectively inhibit AKR1D1 activity in either cell-free or hepatocellular systems. Through manipulation of AKR1D1 expression and activity, we have demonstrated its potent ability to regulate glucocorticoid availability and receptor activation within human hepatoma cells. These data suggest that AKR1D1 may have an important role in regulating endogenous (and potentially exogenous) glucocorticoid action that may be of particular relevance to physiological and pathophysiological processes affecting the liver.


Assuntos
Carcinoma Hepatocelular/metabolismo , Glucocorticoides/metabolismo , Neoplasias Hepáticas/metabolismo , Oxirredutases/metabolismo , Receptores de Glucocorticoides/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Fígado/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-30431220

RESUMO

Histone lysine demethylase (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

5.
J Bone Miner Res ; 33(12): 2091-2098, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30184270

RESUMO

Atypical femoral fractures (AFFs) are a rare but potentially devastating event, often but not always linked to bisphosphonate (BP) therapy. The pathogenic mechanisms underlying AFFs remain obscure, and there are no tests available that might assist in identifying those at high risk of AFF. We previously used exome sequencing to explore the genetic background of three sisters with AFFs and three additional unrelated AFF cases, all previously treated with BPs. We detected 37 rare mutations (in 34 genes) shared by the three sisters. Notably, we found a p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase, a component of the mevalonate pathway, which is critical to osteoclast function and is inhibited by N-BPs. In addition, the CYP1A1 gene, responsible for the hydroxylation of 17ß-estradiol, estrone, and vitamin D, was also mutated in all three sisters and one unrelated patient. Here we present a detailed list of the variants found and report functional analyses of the GGPS1 p.Asp188Tyr mutation, which showed a severe reduction in enzyme activity together with oligomerization defects. Unlike BP treatment, this genetic mutation will affect all cells in the carriers. RNAi knockdown of GGPS1 in osteoblasts produced a strong mineralization reduction and a reduced expression of osteocalcin, osterix, and RANKL, whereas in osteoclasts, it led to a lower resorption activity. Taken together, the impact of the mutated GGPPS and the relevance of the downstream effects in bone cells make it a strong candidate for AFF susceptibility. We speculate that other genes such as CYP1A1 might be involved in AFF pathogenesis, which remains to be functionally proved. The identification of the genetic background for AFFs provides new insights for future development of novel risk assessment tools. © 2018 American Society for Bone and Mineral Research.

6.
J Med Chem ; 61(11): 4883-4903, 2018 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-29767973

RESUMO

Aldehyde dehydrogenases (ALDHs) are responsible for the metabolism of aldehydes (exogenous and endogenous) and possess vital physiological and toxicological functions in areas such as CNS, inflammation, metabolic disorders, and cancers. Overexpression of certain ALDHs (e.g., ALDH1A1) is an important biomarker in cancers and cancer stem cells (CSCs) indicating the potential need for the identification and development of small molecule ALDH inhibitors. Herein, a newly designed series of quinoline-based analogs of ALDH1A1 inhibitors is described. Extensive medicinal chemistry optimization and biological characterization led to the identification of analogs with significantly improved enzymatic and cellular ALDH inhibition. Selected analogs, e.g., 86 (NCT-505) and 91 (NCT-506), demonstrated target engagement in a cellular thermal shift assay (CETSA), inhibited the formation of 3D spheroid cultures of OV-90 cancer cells, and potentiated the cytotoxicity of paclitaxel in SKOV-3-TR, a paclitaxel resistant ovarian cancer cell line. Lead compounds also exhibit high specificity over other ALDH isozymes and unrelated dehydrogenases. The in vitro ADME profiles and pharmacokinetic evaluation of selected analogs are also highlighted.

7.
Chem Commun (Camb) ; 53(99): 13264-13267, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29186216

RESUMO

Histone lysine methylation is regulated by Nε-methyltransferases, demethylases, and Nε-methyl lysine binding proteins. Thermodynamic, catalytic and computational studies were carried out to investigate the interaction of three epigenetic protein classes with synthetic histone substrates containing l- and d-lysine residues. The results reveal that out of the three classes, Nε-methyl lysine binding proteins are superior in accepting lysines with the d-configuration.


Assuntos
Epigênese Genética/genética , Histona Desmetilases/metabolismo , Lisina/química , Metiltransferases/metabolismo , Biocatálise , Histona Desmetilases/genética , Lisina/metabolismo , Metilação , Metiltransferases/genética , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Termodinâmica
9.
J Bone Miner Res ; 32(9): 1860-1869, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28337806

RESUMO

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen-containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1-fluoro-2-(imidazo-[1,2 alpha]pyridin-3-yl)-ethyl-bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short-term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3-NSG murine model of myeloma-induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 American Society for Bone and Mineral Research.


Assuntos
Difosfonatos/farmacologia , Difosfonatos/farmacocinética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley
10.
Bioconjug Chem ; 27(2): 329-40, 2016 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-26646666

RESUMO

A bone imaging toolkit of 21 fluorescent probes with variable spectroscopic properties, bone mineral binding affinities, and antiprenylation activities has been created, including a novel linking strategy. The linking chemistry allows attachment of a diverse selection of dyes fluorescent in the visible to near-infrared range to any of the three clinically important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate, and minodronate or their analogues). The resultant suite of conjugates offers multiple options to "mix and match" parent drug structure, fluorescence emission wavelength, relative bone affinity, and presence or absence of antiprenylation activity, for bone-related imaging applications.


Assuntos
Doenças Ósseas/diagnóstico , Osso e Ossos/patologia , Difosfonatos/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Animais , Linhagem Celular , Humanos , Masculino , Ratos Sprague-Dawley
11.
Bone ; 81: 478-486, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26318908

RESUMO

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.


Assuntos
Difosfonatos/química , Geraniltranstransferase/antagonistas & inibidores , Mutação , Nitrogênio/química , Conservadores da Densidade Óssea/uso terapêutico , Catálise , Domínio Catalítico , Cristalização , Difosfonatos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Geraniltranstransferase/química , Humanos , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Imidazóis/uso terapêutico , Concentração Inibidora 50 , Conformação Molecular , Oligonucleotídeos/química , Ligação Proteica , Proteínas Recombinantes/química , Treonina/química , Tirosina/química , Ácido Zoledrônico
12.
Eur J Med Chem ; 84: 77-89, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25016230

RESUMO

Phosphonocarboxylate (PC) analogs of the anti-osteoporotic drugs, bisphosphonates, represent the first class of selective inhibitors of Rab geranylgeranyl transferase (RabGGTase, RGGT), an enzyme implicated in several diseases including ovarian, breast and skin cancer. Here we present the synthesis and biological characterization of an extended set of this class of compounds, including lipophilic derivatives of the known RGGT inhibitors. From this new panel of PCs, we have identified an inhibitor of RGGT that is of similar potency as the most active published phosphonocarboxylate, but of higher selectivity towards this enzyme compared to prenyl pyrophosphate synthases. New insights into structural requirements are also presented, showing that only PC analogs of the most potent 3rd generation bisphosphonates inhibit RGGT. In addition, the first phosphonocarboxylate-derived GGPPS inhibitor is reported.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Organofosfonatos/farmacologia , Alquil e Aril Transferases/metabolismo , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HeLa , Humanos , Estrutura Molecular , Organofosfonatos/síntese química , Organofosfonatos/química , Relação Estrutura-Atividade
13.
Chem Biol Interact ; 207: 52-7, 2014 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-24246760

RESUMO

Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11ß-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.


Assuntos
Oxirredutases/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Sequência de Aminoácidos , Animais , Benzaldeídos/metabolismo , Western Blotting , Imunofluorescência , Humanos , Membranas Intracelulares/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Microssomos Hepáticos/enzimologia , Dados de Sequência Molecular , NAD/metabolismo , NADP/metabolismo , Nitrosaminas/química , Nitrosaminas/metabolismo , Oxirredutases/química , Células Sf9 , Espectrofotometria , Especificidade por Substrato , Ultracentrifugação
14.
Bone ; 49(1): 20-33, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21497677

RESUMO

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.


Assuntos
Difosfonatos/química , Difosfonatos/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Difosfonatos/metabolismo , Humanos , Modelos Biológicos , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Relação Estrutura-Atividade
15.
Biochem Biophys Res Commun ; 407(4): 663-7, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21420384

RESUMO

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K(isom)) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K(isom) values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway. As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K(isom) calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.


Assuntos
Dimetilaliltranstransferase/antagonistas & inibidores , Difosfonatos/farmacologia , Ácido Mevalônico/metabolismo , Células Cultivadas , Dimetilaliltranstransferase/química , Difosfonatos/química , Humanos , Nitrogênio/química , Fatores de Tempo
16.
J Biol Chem ; 286(5): 3315-22, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21084289

RESUMO

We present here a study of a eukaryotic trans-prenylsynthase from the malaria pathogen Plasmodium vivax. Based on the results of biochemical assays and contrary to previous indications, this enzyme catalyzes the production of geranylgeranyl pyrophosphate (GGPP) rather than farnesyl pyrophosphate (FPP). Structural analysis shows that the product length is constrained by a hydrophobic cavity formed primarily by a set of residues from the same subunit as the product as well as at least one other from the dimeric partner. Furthermore, Plasmodium GGPP synthase (GGPPS) can bind nitrogen-containing bisphosphonates (N-BPs) strongly with the energetically favorable cooperation of three Mg(2+), resulting in inhibition by this class of compounds at IC(50) concentrations below 100 nM. In contrast, human and yeast GGPPSs do not accommodate a third magnesium atom in the same manner, resulting in their insusceptibility to N-BPs. This differentiation is in part attributable to a deviation in a conserved motif known as the second aspartate-rich motif: whereas the aspartates at the start and end of the five-residue motif in FFPP synthases and P. vivax GGPPSs both participate in the coordination of the third Mg(2+), an asparagine is featured as the last residue in human and yeast GGPPSs, resulting in a different manner of interaction with nitrogen-containing ligands.


Assuntos
Geranil-Geranildifosfato Geranil-Geraniltransferase/química , Plasmodium vivax/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Difosfonatos/metabolismo , Difosfonatos/farmacologia , Inibidores Enzimáticos , Geranil-Geranildifosfato Geranil-Geraniltransferase/antagonistas & inibidores , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Magnésio , Nitrogênio , Fosfatos de Poli-Isoprenil/biossíntese , Leveduras
17.
J Med Chem ; 53(9): 3454-64, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-20394422

RESUMO

3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Lactatos/farmacologia , Organofosfonatos/farmacologia , Compostos Organofosforados/farmacologia , Conservadores da Densidade Óssea/síntese química , Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/tratamento farmacológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Ácido Etidrônico/análogos & derivados , Ácido Etidrônico/farmacologia , Ácido Etidrônico/uso terapêutico , Humanos , Lactatos/síntese química , Lactatos/uso terapêutico , Organofosfonatos/síntese química , Organofosfonatos/uso terapêutico , Compostos Organofosforados/síntese química , Compostos Organofosforados/uso terapêutico , Osteoporose/tratamento farmacológico , Prenilação de Proteína/efeitos dos fármacos , Ácido Risedrônico , Estereoisomerismo , Proteínas rab de Ligação ao GTP
18.
PLoS One ; 4(10): e7113, 2009 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-19841672

RESUMO

UNLABELLED: Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.


Assuntos
Oxirredutases do Álcool/química , Aldeído Redutase , Aldo-Ceto Redutases , Antineoplásicos/farmacologia , Clonagem Molecular , Cristalografia por Raios X/métodos , Etanolaminas/química , Humanos , Isoquinolinas/química , Cinética , Mutagênese , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura Ambiente , Xenobióticos/química
19.
Chem Biol ; 15(12): 1296-306, 2008 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-19101474

RESUMO

Cryptosporidiosis is a neglected disease without a wholly effective drug. We present a study demonstrating nitrogen-containing bisphosphonates (N-BPs) to be capable of inhibiting Cryptosporidium parvum at low micromolar concentrations in infected MDCK cells. Predictably, the mechanism of action is based on inhibition of biosynthesis of isoprenoids but the target enzyme is unexpectedly a distinctive C. parvum enzyme dubbed nonspecific polyprenyl pyrophosphate synthase (CpNPPPS). This enzyme produces various isoprenoid products larger than FPP and is inhibited by N-BPs at subnanomolar concentrations. It is part of an isoprenoid pathway in Cryptosporidium distinctly different from other organisms. The proposed mechanism of action is corroborated by crystal structures of the enzyme with risedronate and zoledronate bound showing how this enzyme's unique chain length determinant region enables it to accommodate larger substrates and products. These results, combined with existing data on their clinical use, demonstrate that N-BPs are very promising anticryptosporidial drug candidates.


Assuntos
Anti-Infecciosos/uso terapêutico , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/enzimologia , Dimetilaliltranstransferase/metabolismo , Difosfonatos/uso terapêutico , Animais , Bovinos , Células Cultivadas , Cromatografia Líquida , Cristalografia por Raios X , Dimetilaliltranstransferase/antagonistas & inibidores , Difosfonatos/farmacologia , Imunofluorescência , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Prenilação de Proteína
20.
Endocrinology ; 149(11): 5688-95, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18617622

RESUMO

Clinical studies have shown that total body fat mass is related to both bone density and fracture risk and that fat ingestion reduces bone turnover. These effects are at least partially mediated by endocrine mechanisms, but it is possible that lipids might act directly on bone. We assessed the effects of broad fractions of milk lipids in osteoblasts, bone marrow, and neonatal mouse calvariae. Several milk fractions and their hydrolysates inhibited osteoclastogenesis in bone marrow cultures, so we assessed the effects of free fatty acids in this model. Saturated fatty acids (0.1-10 microg/ml) inhibited osteoclastogenesis in bone marrow cultures and RAW264.7 cells. This effect was maximal for C14:0 to C18:0 fatty acids. The introduction of greater than 1 double bond abrogated this effect; omega3 and omega6 fatty acids had comparable low activity. Osteoblast proliferation was modestly increased by the antiosteoclastogenic compounds, ruling out a nonspecific toxic effect. Active fatty acids did not consistently change expression of receptor activator of nuclear factor-kappaB ligand or osteoprotegerin in osteoblastic cells nor did they affect the activity of key enzymes in the mevalonate pathway. However, receptors known to bind fatty acids were found to be expressed in osteoblastic (GPR120) and osteoclastic (GPR40, 41, 43, 120) cells. A synthetic GPR 40/120 agonist mimicked the inhibitory effects of fatty acids on osteoclastogenesis. These findings provide a novel link between lipid and bone metabolism, which might contribute to the positive relationship between adiposity and bone density as well as provide novel targets for pharmaceutical and nutriceutical development.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácidos Graxos/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Osso e Ossos/citologia , Diferenciação Celular/genética , Células Cultivadas , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/fisiologia , Masculino , Ácido Mevalônico/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Ratos , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo
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