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1.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

2.
Trends Pharmacol Sci ; 42(6): 431-433, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33867130

RESUMO

Latest research shows that SERPINE1 overexpression has an important role in Coronavirus 2019 (COVID-19)-associated coagulopathy leading to acute respiratory distress syndrome (ARDS). However, ways to target this protein remain elusive. In this forum, we discuss recent evidence linking SERPINE1 with COVID-19-related ARDS and summarize the available data on inhibitors of this target.


Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Ativação do Complemento , Humanos , SARS-CoV-2
3.
Drug Discov Today ; 26(2): 442-454, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33259801

RESUMO

Serine protease inhibitors (serpins) are a large family of proteins that regulate and control crucial physiological processes, such as inflammation, coagulation, thrombosis and thrombolysis, and immune responses. The extraordinary impact that these proteins have on numerous crucial pathways makes them an attractive target for drug discovery. In this review, we discuss recent advances in research on small-molecule modulators of serpins, examine their mode of action, analyse the structural data from crystallised protein-ligand complexes, and highlight the potential obstacles and possible therapeutic perspectives. The application of in silico methods for rational drug discovery is also summarised. In addition, we stress the need for continued research in this field.

4.
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 , Ligação 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
5.
PLoS One ; 10(2): e0117470, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25658953

RESUMO

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3' to 5' direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25-47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.


Assuntos
Proteínas de Bactérias/química , Exodesoxirribonucleases/química , Methylococcaceae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Reparo do DNA/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Temperatura Alta , Methylococcaceae/genética
6.
BMC Cell Biol ; 16: 1, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25655076

RESUMO

BACKGROUND: EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however the precise mechanism of substrate recognition and sorting to the ERAD pathway is still poorly understood. It has previously been demonstrated that EDEM1 and EDEM2 binding does not require the trimming of substrate glycans or even ERAD substrate glycosylation, thus suggesting that both chaperones probably recognize misfolded regions of aberrant proteins. RESULTS: In this work, we focused on the substrate recognition by EDEM1 and EDEM2, asking whether hydrophobicity of protein determinants might be important for these interactions in human cells. In the study we used ricin, a protein toxin that utilizes the ERAD pathway in its retrotranslocation from the ER to the cytosol, and a model misfolded protein, the pancreatic isoform of human ß-secretase, BACE457. Mutations in the hydrophobic regions of these proteins allowed us to obtain mutated forms with increased and decreased hydrophobicity. CONCLUSIONS: Our data provide the first evidence that recognition of ERAD substrates by EDEM1 and EDEM2 might be determined by a sufficiently high hydrophobicity of protein determinants. Moreover, EDEM proteins can bind hydrophobic transmembrane regions of misfolded ERAD substrates. These data contribute to the general understanding of the regulation of ERAD in mammalian cells.


Assuntos
Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , alfa-Manosidase/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Dicroísmo Circular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Ricina/química , Ricina/genética , Ricina/metabolismo , Especificidade por Substrato
7.
Drug Discov Today ; 20(6): 743-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25555749

RESUMO

The Jumonji (Jmj) family of demethylases has a crucial role in regulating epigenetic processes through the removal of methyl groups from histone tails. The ability of Jmj demethylases to recognise their targets selectively has been elegantly addressed by structural studies. Reviewing recent structural literature, we provide an overview of selectivity mechanisms that demethylases use, including specific residues, methylation states and contextual requirements. We also report the presence of a common JmjN support domain across the family. The ability to use structural information for this enzyme class will be a crucial component of future drug discovery.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/química , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
8.
Nature ; 510(7505): 422-426, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24814345

RESUMO

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.


Assuntos
Eucariotos/enzimologia , Modelos Moleculares , Oxigenases/química , Células Procarióticas/enzimologia , Ribossomos/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Sequência Conservada , Eucariotos/classificação , Humanos , Oxigenases/metabolismo , Filogenia , Células Procarióticas/classificação , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência
9.
Structure ; 21(7): 1182-92, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23791943

RESUMO

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.


Assuntos
Proteínas de Bactérias/química , Carboxiliases/química , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Carboxiliases/deficiência , Carboxiliases/genética , Domínio Catalítico , Cristalografia por Raios X , Deficiências Nutricionais/genética , Estabilidade Enzimática , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína
10.
Biochem J ; 452(1): 27-36, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23425511

RESUMO

MAT (methionine adenosyltransferase) utilizes L-methionine and ATP to form SAM (S-adenosylmethionine), the principal methyl donor in biological methylation. Mammals encode a liver-specific isoenzyme, MAT1A, that is genetically linked with an inborn metabolic disorder of hypermethioninaemia, as well as a ubiquitously expressed isoenzyme, MAT2A, whose enzymatic activity is regulated by an associated subunit MAT2B. To understand the molecular mechanism of MAT functions and interactions, we have crystallized the ligand-bound complexes of human MAT1A, MAT2A and MAT2B. The structures of MAT1A and MAT2A in binary complexes with their product SAM allow for a comparison with the Escherichia coli and rat structures. This facilitates the understanding of the different substrate or product conformations, mediated by the neighbouring gating loop, which can be accommodated by the compact active site during catalysis. The structure of MAT2B reveals an SDR (short-chain dehydrogenase/reductase) core with specificity for the NADP/H cofactor, and harbours the SDR catalytic triad (YxxxKS). Extended from the MAT2B core is a second domain with homology with an SDR sub-family that binds nucleotide-sugar substrates, although the equivalent region in MAT2B presents a more open and extended surface which may endow a different ligand/protein-binding capability. Together, the results of the present study provide a framework to assign structural features to the functional and catalytic properties of the human MAT proteins, and facilitate future studies to probe new catalytic and binding functions.


Assuntos
Domínio Catalítico , Cristalização , Metionina Adenosiltransferase/química , S-Adenosilmetionina/biossíntese , Animais , Domínio Catalítico/fisiologia , Cristalização/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/fisiologia , Fígado/enzimologia , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/fisiologia , Mapeamento de Interação de Proteínas/métodos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Ratos , S-Adenosilmetionina/química , Especificidade por Substrato/fisiologia
11.
BMC Struct Biol ; 12: 14, 2012 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-22720794

RESUMO

BACKGROUND: Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. RESULTS: The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-ß-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. CONCLUSIONS: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.


Assuntos
Biocatálise , Glutamil Aminopeptidase/química , Família Multigênica , Sequência de Aminoácidos , Bactérias/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Humanos , Metais , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Especificidade por Substrato
12.
Biochem Biophys Res Commun ; 419(3): 485-9, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22366092

RESUMO

Zn(2+)-dependent carbonic anhydrases (CA) catalyse the reversible hydration of carbon dioxide to bicarbonate and participate in diverse physiological processes, hence having manifold therapeutic potentials. Among the 15 human CAs with wide-ranging sub-cellular localisation and kinetic properties, CA VI is the only secretory isoform. The 1.9Å crystal structure of the human CA VI catalytic domain reveals a prototypical mammalian CA fold, and a novel dimeric arrangement as compared to previously-reported CA structures. The active site cavity contains a cluster of non-conserved residues that may be involved in ligand binding and have significant implications for developing the next-generation of isoform-specific inhibitors.


Assuntos
Anidrases Carbônicas/química , Descoberta de Drogas , Inibidores Enzimáticos/química , Zinco/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Isoenzimas/química , Conformação Proteica , Multimerização Proteica
13.
PLoS One ; 5(11): e13719, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-21072165

RESUMO

BACKGROUND: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. PRINCIPAL FINDINGS: To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. CONCLUSIONS: Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. 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 S2.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/química , Sítios de Ligação , Ligação Competitiva , Biocatálise/efeitos dos fármacos , Cristalografia por Raios X , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , NAD/química , NAD/metabolismo , Oxirredução , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
14.
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 , Xenobióticos/química
16.
J Mol Biol ; 388(5): 997-1008, 2009 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-19345228

RESUMO

Acyl-CoA synthetases belong to the superfamily of adenylate-forming enzymes, and catalyze the two-step activation of fatty acids or carboxylate-containing xenobiotics. The carboxylate substrate first reacts with ATP to form an acyl-adenylate intermediate, which then reacts with CoA to produce an acyl-CoA ester. Here, we report the first crystal structure of a medium-chain acyl-CoA synthetase ACSM2A, in a series of substrate/product/cofactor complexes central to the catalytic mechanism. We observed a substantial rearrangement between the N- and C-terminal domains, driven purely by the identity of the bound ligand in the active site. Our structures allowed us to identify the presence or absence of the ATP pyrophosphates as the conformational switch, and elucidated new mechanistic details, including the role of invariant Lys557 and a divalent magnesium ion in coordinating the ATP pyrophosphates, as well as the involvement of a Gly-rich P-loop and the conserved Arg472-Glu365 salt bridge in the domain rearrangement.


Assuntos
Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Conformação Proteica , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Coenzima A/metabolismo , Coenzima A Ligases/genética , Cristalografia por Raios X , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por Substrato
17.
Bioorg Med Chem ; 17(2): 530-6, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19097799

RESUMO

In order to extend the chemical diversity available for organic polyketide synthesis, the concept of propionate scanning was developed. We observed that naturally occurring polyketides frequently comprise not only acetate, but also some propionate as building blocks. Therefore our approach consists of a systematic replacement of some of the acetate building blocks during synthesis by propionate moieties, resulting in additional methyl groups that may give rise to different properties of the polyketides. Here we present the results of a first 'proof of concept' study where a novel zearalenone analogue 5 was prepared that comprises an additional methyl group at C5'. Key steps in the synthesis of 5 include a Marshall-Tamaru reaction, a Suzuki cross-coupling reaction, and a Mitsunobu lactonization. Compared to the parent zearalenone (1), analogue 5 showed reduced binding to a panel of human protein kinases and no binding to human Hsp90. On the other hand, however, 5 turned out to be a potent (IC(50)=210 nM) inhibitor of human carbonyl reductase 1 (CBR1).


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Macrolídeos/química , Propionatos/química , Zearalenona/química , Acetatos , Estrogênios não Esteroides , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Macrolídeos/farmacologia , Ligação Proteica , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Zearalenona/farmacologia
18.
Chem Biol Interact ; 178(1-3): 234-41, 2009 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-19061875

RESUMO

Human carbonyl reductase is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily and is known to play an important role in the detoxification of xenobiotics bearing a carbonyl group. The two monomeric NADPH-dependent human isoforms of cytosolic carbonyl reductase CBR1 and CBR3 show a sequence similarity of 85% on the amino acid level, which is definitely high if compared to the low similarities usually observed among other members of the SDR superfamily (15-30%). Despite the sequence similarity and the similar features found in the available crystal structures of the two enzymes, CBR3 shows only low or no activity towards substrates that are metabolised by CBR1. This surprising substrate specificity is still not fully understood. In the present study, we introduced several point mutations and changed sequences of up to 17 amino acids of CBR3 to the corresponding amino acids of CBR1, to gather insight into the catalytic mechanism of both enzymes. Proteins were expressed in Escherichia coli and purified by Ni-affinity chromatography. Their catalytic properties were then compared using isatin and 9,10-phenanthrenequinone as model substrates. Towards isatin, wild-type CBR3 showed a catalytic efficiency of 0.018 microM(-1)min(-1), whereas wild-type CBR1 showed a catalytic efficiency of 13.5 microM(-1)min(-1). In particular, when nine residues (236-244) in the vicinity of the catalytic center and a proline (P230) in CBR3 were mutated to the corresponding residues of CBR1 a much higher k(cat)/K(m) value (5.7 microM(-1)min(-1)) towards isatin was observed. To gain further insight into the protein-ligand binding process, docking simulations were perfomed on this mutant and on both wild-type enzymes (CBR1 and CBR3). The theoretical model of the mutant was ad hoc built by means of standard comparative modelling.


Assuntos
Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Biocatálise , Primers do DNA , DNA Complementar , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
19.
Nature ; 448(7149): 87-91, 2007 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-17589501

RESUMO

Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.


Assuntos
Proteínas de Ligação a DNA/química , Histonas/metabolismo , Oxirredutases N-Desmetilantes/química , Fatores de Transcrição/química , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases com o Domínio Jumonji , Modelos Moleculares , Oxirredutases N-Desmetilantes/metabolismo , Conformação Proteica , Proteínas Recombinantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Transcrição/metabolismo
20.
FEBS Lett ; 580(1): 273-7, 2006 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-16376343

RESUMO

Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of beta-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation.


Assuntos
Adesinas Bacterianas/química , Fibronectinas/química , Peptídeos/química , Staphylococcus aureus/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Secundária de Proteína , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade
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