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1.
Sci Rep ; 14(1): 2178, 2024 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-38272944

RESUMO

Recognition of the mRNA 5' end is a critical step needed for translation initiation. This step is performed by the cap binding protein eIF4E, which joins the larger eIF4G subunit to form the eIF4F complex. Trypanosomatids have a minimum of five different eIF4F-like complexes formed through specific but not well-defined interactions between four different eIF4E and five eIF4G homologues. The EIF4E6/EIF4G5 complex has been linked with the stage-specific translation of mRNAs encoding the major Trypanosoma brucei virulence factors. Here, to better define the molecular basis for the TbEIF4E6/TbEIF4G5 interaction, we describe the identification of the peptide interacting with TbEIF4E6 in the region comprising residues 79-166 of TbEIF4G5. The TbEIF4E6-TbEIF4G5_79-116 complex reconstituted with recombinant proteins is highly stable even in the absence of cap-4. The crystal structure of the complex was subsequently solved, revealing extensive interacting surfaces. Comparative analyses highlight the conservation of the overall structural arrangement of different eIF4E/eIF4G complexes. However, highly different interacting surfaces are formed with distinct binding contacts occurring both in the canonical and noncanonical elements within eIF4G and the respective eIF4E counterpart. These specific pairs of complementary interacting surfaces are likely responsible for the selective association needed for the formation of distinct eIF4F complexes in trypanosomatids.


Assuntos
Fator de Iniciação 4F em Eucariotos , Trypanosoma brucei brucei , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Trypanosoma brucei brucei/genética , Ligação Proteica , RNA Mensageiro/metabolismo
2.
Eur J Med Chem ; 246: 114941, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36455355

RESUMO

Nicotinamide adenine dinucleotide kinases (NAD kinases) are essential and ubiquitous enzymes involved in the production of NADP(H) which is an essential cofactor in many metabolic pathways. Targeting NAD kinase (NADK), a rate limiting enzyme of NADP biosynthesis pathway, represents a new promising approach to treat bacterial infections. Previously, we have produced the first NADK inhibitor active against staphylococcal infection. From this linear di-adenosine derivative, namely NKI1, we designed macrocyclic analogues. Here, we describe the synthesis and evaluation of an original series of cyclic diadenosine derivatives as NADK inhibitors of two pathogenic bacteria, Listeria monocytogenes and Staphylococcus aureus. The nature and length of the link between the two adenosine units were examined leading to sub-micromolar inhibitors of NADK1 from L. monocytogenes, including its most potent in vitro inhibitor reported so far (with a 300-fold improvement compared to NKI1).


Assuntos
Adenosina , Fosfotransferases (Aceptor do Grupo Álcool) , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adenosina/farmacologia , Relação Estrutura-Atividade , Bactérias/metabolismo
3.
FEBS J ; 290(2): 482-501, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36036789

RESUMO

Multidrug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to the failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+ -competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here, we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 µm. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model.


Assuntos
Antibacterianos , NAD , Pseudomonas aeruginosa , Animais , Antibacterianos/farmacologia , Antibacterianos/química , NAD/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool) , Pseudomonas aeruginosa/efeitos dos fármacos , Peixe-Zebra , Desenho de Fármacos
4.
Science ; 372(6541): 520-524, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33926956

RESUMO

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution.


Assuntos
2-Aminopurina/análogos & derivados , Replicação do DNA , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/química , Polimerização , Siphoviridae/química , Siphoviridae/enzimologia , Proteínas não Estruturais Virais/química , 2-Aminopurina/química , DNA Polimerase Dirigida por DNA/classificação , DNA Polimerase Dirigida por DNA/genética , Genoma Viral , Filogenia , Siphoviridae/genética , Proteínas não Estruturais Virais/classificação , Proteínas não Estruturais Virais/genética
5.
Molecules ; 25(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105870

RESUMO

Nicotinamide adenine dinucleotide (NAD) kinases are essential and ubiquitous enzymes involved in the tight regulation of NAD/nicotinamide adenine dinucleotide phosphate (NADP) levels in many metabolic pathways. Consequently, they represent promising therapeutic targets in cancer and antibacterial treatments. We previously reported diadenosine derivatives as NAD kinase inhibitors with bactericidal activities on Staphylococcus aureus. Among them, one compound (namely NKI1) was found effective in vivo in a mouse infection model. With the aim to gain detailed knowledge about the selectivity and mechanism of action of this lead compound, we planned to develop a chemical probe that could be used in affinity-based chemoproteomic approaches. Here, we describe the first functionalized chemical probe targeting a bacterial NAD kinase. Aminoalkyl functional groups were introduced on NKI1 for further covalent coupling to an activated SepharoseTM matrix. Inhibitory properties of functionalized NKI1 derivatives together with X-ray characterization of their complexes with the NAD kinase led to identify candidate compounds that are amenable to covalent coupling to a matrix.


Assuntos
Adenina/análogos & derivados , Adenosina/síntese química , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Adenina/síntese química , Adenina/farmacologia , Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Camundongos , Modelos Moleculares , NADP/química , Conformação Proteica , Sefarose/química , Staphylococcus aureus
6.
ACS Infect Dis ; 6(3): 422-435, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32017533

RESUMO

Antibiotic resistance is a worldwide threat due to the decreasing supply of new antimicrobials. Novel targets and innovative strategies are urgently needed to generate pathbreaking drug compounds. NAD kinase (NADK) is essential for growth in most bacteria, as it supports critical metabolic pathways. Here, we report the discovery of a new class of antibacterials that targets bacterial NADK. We generated a series of small synthetic adenine derivatives to screen those harboring promising substituents in order to guide efficient fragment linking. This led to NKI1, a new lead compound inhibiting NADK that showed in vitro bactericidal activity against Staphylococcus aureus. In a murine model of infection, NKI1 restricted survival of the bacteria, including methicillin-resistant S. aureus. Collectively, these findings identify bacterial NADK as a potential drug target and NKI1 as a lead compound in the treatment of staphylococcal infections.


Assuntos
Antibacterianos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Adenina/química , Adenina/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Feminino , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
7.
Chembiochem ; 21(10): 1412-1417, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31899839

RESUMO

The structurally unique "fleximer" nucleosides were originally designed to investigate how flexibility in a nucleobase could potentially affect receptor-ligand recognition and function. Recently they have been shown to have low-to-sub-micromolar levels of activity against a number of viruses, including coronaviruses, filoviruses, and flaviviruses. However, the synthesis of distal fleximers in particular has thus far been quite tedious and low yielding. As a potential solution to this issue, a series of proximal fleximer bases (flex-bases) has been successfully coupled to both ribose and 2'-deoxyribose sugars by using the N-deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purine nucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on a thieno-expanded tricyclic heterocyclic base, as well as several distal and proximal flex-bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion, thus potentially significantly shortening the route to these biologically significant compounds. The results of those studies are reported herein.


Assuntos
Escherichia coli/enzimologia , Lactobacillus leichmannii/enzimologia , Nucleosídeos/biossíntese , Pentosiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Glicosilação , Estrutura Molecular
8.
Nucleic Acids Res ; 47(11): 5973-5987, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31066441

RESUMO

Association of the initiation factor eIF4E with the mRNA cap structure is a key step for translation. Trypanosomatids present six eIF4E homologues, showing a low conservation and also differing significantly from the IF4Es of multicellular eukaryotes. On the mRNA side, while in most eukaryotes the mRNA contains cap-0 (7-methyl-GTP), the trypanosomatid mRNA features a cap-4, which is formed by a cap-0, followed by the AACU sequence containing 2'-O-ribose methylations and base methylations on nucleotides 1 and 4. The studies on eIF4E-cap-4 interaction have been hindered by the difficulty to synthesize this rather elaborated cap-4 sequence. To overcome this problem, we applied a liquid-phase oligonucleotide synthesis strategy and describe for the first time the crystal structure of a trypanosomatid eIF4E (T. cruzi EIF4E5) in complex with cap-4. The TcEIF4E5-cap-4 structure allowed a detailed description of the binding mechanism, revealing the interaction mode for the AACU sequence, with the bases packed in a parallel stacking conformation and involved, together with the methyl groups, in hydrophobic contacts with the protein. This binding mechanism evidences a distinct cap interaction mode in comparison with previously described eIF4E structures and may account for the difference of TcEIF4E5-cap-4 dissociation constant in comparison with other eIF4E homologues.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Capuzes de RNA/química , Trypanosoma cruzi/química , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Metilação de DNA , Humanos , Ligantes , Modelos Moleculares , Nucleotídeos/química , Oligonucleotídeos , Ligação Proteica , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/metabolismo , Schistosoma mansoni/metabolismo , Temperatura , Trypanosoma/metabolismo
9.
Org Biomol Chem ; 17(2): 290-301, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30543241

RESUMO

We developed a versatile access to a series of 4-substituted imidazole 2'-deoxynucleoside triphosphate bearing functionalized phenyl or pyrimidinyl rings. 4-Iodo-1H-imidazole was enzymatically converted into the corresponding 2'-deoxynucleoside, which was then chemically derived into its 5'-triphosphate, followed by 4-arylation via Suzuki-Miyaura coupling using (hetero)arylboronic acids. Both KF (exo-) and Deep Vent (exo-) DNA polymerases incorporated these modified nucleotides in primer-extension assays, adenine being the preferred pairing partner in the template. The 4-(3-aminophenyl)imidazole derivative (3APh) was the most efficiently inserted opposite A by KF (exo-) with only a 37-fold lower efficiency (Vmax/KM) than that of the correct dTTP. No further extension occurred after the incorporation of a single aryl-imidazole nucleotide. Interestingly, the aryl-imidazole dNTPs were found to undergo successive incorporation by calf thymus terminal deoxynucleotidyl transferase with different tailing efficiencies among this series and with a marked preference for 2APyr polymerization.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleosídeos/metabolismo , Imidazóis/metabolismo , Polifosfatos/metabolismo , Pirimidinas/metabolismo , Animais , Sequência de Bases , Bovinos , DNA Polimerase I/metabolismo , Desoxirribonucleosídeos/síntese química , Desoxirribonucleosídeos/química , Imidazóis/síntese química , Imidazóis/química , Polimerização , Polifosfatos/síntese química , Polifosfatos/química , Pirimidinas/síntese química , Pirimidinas/química
10.
Nucleic Acids Res ; 46(12): 6271-6284, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29788485

RESUMO

Nucleic acid aptamers, especially RNA, exhibit valuable advantages compared to protein therapeutics in terms of size, affinity and specificity. However, the synthesis of libraries of large random RNAs is still difficult and expensive. The engineering of polymerases able to directly generate these libraries has the potential to replace the chemical synthesis approach. Here, we start with a DNA polymerase that already displays a significant template-free nucleotidyltransferase activity, human DNA polymerase theta, and we mutate it based on the knowledge of its three-dimensional structure as well as previous mutational studies on members of the same polA family. One mutant exhibited a high tolerance towards ribonucleotides (NTPs) and displayed an efficient ribonucleotidyltransferase activity that resulted in the assembly of long RNA polymers. HPLC analysis and RNA sequencing of the products were used to quantify the incorporation of the four NTPs as a function of initial NTP concentrations and established the randomness of each generated nucleic acid sequence. The same mutant revealed a propensity to accept other modified nucleotides and to extend them in long fragments. Hence, this mutant can deliver random natural and modified RNA polymers libraries ready to use for SELEX, with custom lengths and balanced or unbalanced ratios.


Assuntos
Aptâmeros de Nucleotídeos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , RNA/biossíntese , DNA Polimerase Dirigida por DNA/química , Humanos , Mutação , Nucleotídeos/metabolismo , Ribonucleotídeos/metabolismo , DNA Polimerase teta
11.
Food Chem ; 259: 36-45, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29680060

RESUMO

Lactoferrin is an iron-binding cationic glycoprotein (pI = 8.7) beneficial for mammal health, especially udder and milk preservation. A new simple two-step method of quantification was developed. Lactoferrin in 1 mL of bovine skim milk was first adsorbed onto 100 mg of macroporous sulfonated-resin at pH 6.8 by rotary stirring for 90 min at 20-25 °C. After washing the resin, lactoferrin was desorbed using 1 mL of 2 M NaCl containing phenylalanine as a dilution marker, then fully resolved and quantified by RP-HPLC at 220 nm using a wide-bore C4 silica column. This robust, inexpensive and flexible method improves selectivity (no protein interference) and sensitivity compared to previous HPLC methods. In-laboratory validation demonstrated its linearity (25 to 514 µg Lf mL-1), accuracy (110 to 98% recovery), and precision (<4%), which were comparable to immuno-based methods. The results for individual raw cow's milk were strongly correlated with results using an ELISA test.


Assuntos
Fracionamento Químico/métodos , Análise de Alimentos/métodos , Lactoferrina/análise , Leite/química , Adsorção , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Calefação , Resinas de Troca Iônica
12.
Eur J Med Chem ; 124: 1041-1056, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27783975

RESUMO

Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.


Assuntos
Adenosina/química , Adenosina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Listeria monocytogenes/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adenosina/metabolismo , Sequência de Aminoácidos , Inibidores Enzimáticos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Conformação Proteica , Ribose/química , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
13.
Eur J Med Chem ; 85: 418-37, 2014 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-25108359

RESUMO

The 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) has been proposed as a new molecular target for cancer treatment. Here, we describe the synthesis of a series of novel 6-aryl- and 6-heteroarylpurine riboside 5'-monophosphates via Suzuki-Miyaura cross-coupling reactions, and their ability to inhibit recombinant rat and human DNPH1. Enzymatic inhibition studies revealed competitive inhibitors in the low micromolar range. Crystal structures of human and rat DNPH1 in complex with one nucleotide from this series, the 6-naphthylpurine derivative, provided detailed structural information, in particular regarding the possible conformations of a long and flexible loop wrapping around the large hydrophobic substituent. Taking advantage of these high-resolution structures, we performed virtual docking studies in order to evaluate enzyme-inhibitor interactions for the whole compound series. Among the synthesized compounds, several molecules exhibited significant in vitro cytotoxicity against human colon cancer (HCT15, HCT116) and human promyelocytic leukemia (HL60) cell lines with IC50 values in the low micromolar range, which correlated with in vitro DNPH1 inhibitory potency.


Assuntos
Desenho de Fármacos , Terapia de Alvo Molecular , N-Glicosil Hidrolases/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Nucleotídeos de Purina/síntese química , Nucleotídeos de Purina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Química Sintética , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Nucleotídeos de Purina/química , Nucleotídeos de Purina/metabolismo , Ratos , Relação Estrutura-Atividade
14.
PLoS One ; 8(11): e80755, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24260472

RESUMO

The gene dnph1 (or rcl) encodes a hydrolase that cleaves the 2'-deoxyribonucleoside 5'-monophosphate (dNMP) N-glycosidic bond to yield a free nucleobase and 2-deoxyribose 5-phosphate. Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N (6)-substituted AMPs, and among them, cytotoxic cytokinin riboside 5'-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth.


Assuntos
Monofosfato de Adenosina/farmacologia , N-Glicosil Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Ratos , Alinhamento de Sequência , Termodinâmica
15.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 2): 247-55, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23385460

RESUMO

Rcl is a novel N-glycoside hydrolase found in mammals that shows specificity for the hydrolysis of 5'-monophosphate nucleotides. Its role in nucleotide catabolism and the resulting production of 2-deoxyribose 5-phosphate has suggested that it might fuel cancer growth. Its expression is regulated by c-Myc, but its role as an oncoprotein remains to be clarified. In parallel, various nucleosides have been shown to acquire pro-apoptotic properties upon 5'-monophosphorylation in cells. These include triciribine, a tricyclic nucleoside analogue that is currently in clinical trials in combination with a farnesyltransferase inhibitor. Similarly, an N(6)-alkyl-AMP has been shown to be cytotoxic. Interestingly, Rcl has been shown to be inhibited by such compounds in vitro. In order to gain better insight into the precise ligand-recognition determinants, the crystallization of Rcl with these nucleotide analogues was attempted. The first crystal structure of Rcl was solved by molecular replacement using its NMR structure in combination with distantly related crystal structures. The structures of Rcl bound to two other nucleotides were then solved by molecular replacement using the previous crystal structure as a template. The resulting structures, solved at high resolution, led to a clear characterization of the protein-ligand interactions that will guide further rational drug design.


Assuntos
N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Nucleotídeos/química , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Acenaftenos/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Cristalização , Ligantes , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Nucleotídeos/genética , Proteínas Oncogênicas/genética , Organofosfonatos/química , Fosforilação , Ligação Proteica/genética , Mapeamento de Interação de Proteínas/métodos , Ratos , Ribonucleotídeos/química , Ribonucleotídeos/genética , Tionucleotídeos/química , Tionucleotídeos/genética , Timidina/análogos & derivados , Timidina/química , Timidina/genética , Difração de Raios X
16.
Structure ; 20(6): 1107-17, 2012 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-22608967

RESUMO

Making new ligands for a given protein by in situ ligation of building blocks (or fragments) is an attractive method. However, it suffers from inherent limitations, such as the limited number of available chemical reactions and the low information content of usual chemical library deconvolution. Here, we describe a focused screening of adenosine derivatives using X-ray crystallography. We discovered an unexpected and biocompatible chemical reactivity and have simultaneously identified the mode of binding of the resulting products. We observed that the NAD kinase from Listeria monocytogenes (LmNADK1) can promote amide formation between 5'-amino-5'-deoxyadenosine and carboxylic acid groups. This unexpected reactivity allowed us to bridge in situ two adenosine derivatives to fully occupy the active NAD site. This guided the design of a close analog showing micromolar inhibition of two human pathogenic NAD kinases and potent bactericidal activity against Staphylococcus aureus in vitro.


Assuntos
Adenosina/análogos & derivados , Antibacterianos/síntese química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Staphylococcus aureus/efeitos dos fármacos , Adenosina/síntese química , Adenosina/química , Adenosina/farmacologia , Motivos de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Biocatálise , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Listeria monocytogenes/enzimologia , Modelos Moleculares , Ligação Proteica , Staphylococcus aureus/crescimento & desenvolvimento
17.
Food Microbiol ; 30(1): 74-82, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22265286

RESUMO

The impact of Gram-negative bacteria on sensory characteristics and production of volatile compounds as well as biogenic amines (BA) in the core of an uncooked pressed type model cheese was investigated in the presence of a defined complex microbial consortium. Eleven strains of Gram-negative bacteria, selected on the basis of their biodiversity and in vitro BA-production ability, were individually tested in a model cheese. Four out of 6 strains of Enterobacteriaceae (Citrobacter freundii UCMA 4217, Klebsiella oxytoca 927, Hafnia alvei B16 and Proteus vulgaris UCMA 3780) reached counts close to 6 log CFU g⁻¹ in the model cheese. In core of cheeses inoculated with Gram-negative bacteria, only slight differences were observed for microbial counts (Enterococcus faecalis or Lactobacillus plantarum count differences below 1 log CFU g⁻¹), acetate concentration (differences below 200 mg kg⁻¹) and texture (greater firmness) in comparison to control cheeses. Cheese core colour, odour and volatile compound composition were not modified. Although ornithine, the precursor of putrescine, was present in all cheeses, putrescine was only detected in cheeses inoculated with H. alvei B16 and never exceeded 2.18 mmol kg⁻¹ cheese dry matter. Cadaverine was only detected in cheeses inoculated with H. alvei B16, K. oxytoca 927, Halomonas venusta 4C1A or Morganella morganii 3A2A but at lower concentrations (<1.05 mmol kg⁻¹ cheese dry matter), although lysine was available. Only insignificant amounts of the detrimental BA histamine and tyramine, as well as isopentylamine, tryptamine or phenylethylamine, were produced in the cheese model by any of the Gram-negative strains, including those which produced these BA at high levels in vitro.


Assuntos
Aminas Biogênicas/análise , Queijo/microbiologia , Manipulação de Alimentos/métodos , Microbiologia de Alimentos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Consórcios Microbianos , Cadaverina/biossíntese , Contagem de Colônia Microbiana , Comportamento do Consumidor , Contaminação de Alimentos , Cinética , Putrescina/biossíntese , Paladar , Compostos Orgânicos Voláteis/análise
18.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 9): 747-55, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21904027

RESUMO

X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed. Crystallization drops are directly exposed to an X-ray beam after cocrystallization or soaking with the desired ligands. The use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection. Three proteins currently under study in our laboratory for ligand screening by X-ray crystallography were used as validation test cases. The protein crystals belonged to different space groups, including a challenging monoclinic case. The resulting diffraction data can lead to clear ligand recognition, including indication of alternating conformations. These results demonstrate a possible method for automation of ligand screening by X-ray crystallography.


Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Difração de Raios X/métodos , Desenho de Fármacos
19.
J Biol Chem ; 285(53): 41806-14, 2010 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-20962348

RESUMO

Rcl is a potential anti-angiogenic therapeutic target that hydrolyzes the N-glycosidic bond of 2'-deoxyribonucleoside 5'-monophosphate, yielding 2-deoxyribose 5-phosphate and the corresponding base. Its recently elucidated solution structure provided the first insight into the molecular basis for the substrate recognition. To facilitate the development of potent and specific inhibitors of Rcl, the active site was probed by site-directed mutagenesis and by the use of substrate analogs. The nucleobase shows weak interactions with the protein, and the deoxyribose binding pocket includes the catalytic triad Tyr-13, Asp-69, and Glu-93 and the phosphate binding site Ser-87 and Ser-117. The phosphomimetic mutation of Ser-17 to Glu prevents substrate binding and, thus, abolishes the activity of Rcl. The synthetic ligand-based analysis of the Rcl binding site shows that substitutions at positions 2 and 6 of the nucleobase as well as large heterocycles are well tolerated. The phosphate group at position 5 of the (deoxy)ribose moiety is the critical binding determinant. This study provides the roadmap for the design of small molecules inhibitors with pharmacological properties.


Assuntos
N-Glicosil Hidrolases/química , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas/química , Alanina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Domínio Catalítico , Ácido Glutâmico/química , Cinética , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Serina/química
20.
Appl Environ Microbiol ; 76(16): 5570-6, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20581187

RESUMO

A multiplex PCR method, aimed at the detection of genes associated with biogenic amine production, identified the odc gene encoding ornithine decarboxylase in 1 of 15 strains of Staphylococcus epidermidis. The ability of the positive strain, S. epidermidis 2015B, to produce putrescine in vitro was demonstrated by high-performance liquid chromatography (HPLC). In this strain, the odc gene was detected on plasmid DNA, suggesting that the ability to form putrescine is carried by a mobile element, which explains the fact that the trait is strain dependent within the S. epidermidis species. A 6,292-bp nucleotide sequence harboring the putative odc gene was determined. S. epidermidis ornithine decarboxylase (ODC) showed 60 to 65% sequence identity with known ODCs of Gram-positive as well as Gram-negative bacteria. Downstream of the odc gene, a gene encoding a putative amino acid transporter was found that shared 59% sequence identity with the ornithine/putrescine exchanger (PotE) of Escherichia coli. Cloning and expression of the potE gene of S. epidermis 2015B in Lactococcus lactis demonstrated that the gene product transported ornithine and putrescine into the cells and efficiently exchanged putrescine for ornithine. Analysis of the flanking regions showed high identity levels with different S. epidermidis plasmid sequences, which would confirm the plasmidic location of the odc operon. It follows that the odc and potE gene pair encodes a putrescine-producing pathway in S. epidermis 2015B that was acquired through horizontal gene transfer.


Assuntos
Putrescina/metabolismo , Staphylococcus epidermidis/metabolismo , Antiporters/genética , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Escherichia coli/genética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Dados de Sequência Molecular , Ornitina/metabolismo , Ornitina Descarboxilase/genética , Plasmídeos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Staphylococcus epidermidis/genética
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