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1.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360555

RESUMO

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Bioensaio/métodos , Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirazinamida/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/isolamento & purificação , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica
2.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578647

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Infecciosos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Animais , Anti-Infecciosos/química , Sítios de Ligação/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Terapia de Alvo Molecular
3.
Eur J Med Chem ; 211: 113021, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33248851

RESUMO

Leucyl-tRNA synthetase (LeuRS) is a clinically validated target for the development of antimicrobials. This enzyme catalyzes the formation of charged tRNALeu molecules, an essential substrate for protein translation. In the first step of catalysis LeuRS activates leucine using ATP, forming a leucyl-adenylate intermediate. Bi-substrate inhibitors that mimic this chemically labile phosphoanhydride-linked nucleoside have proven to be potent inhibitors of different members of the aminoacyl-tRNA synthetase family but, to date, they have demonstrated poor antibacterial activity. We synthesized a small series of 1,5-anhydrohexitol-based analogues coupled to a variety of triazoles and performed detailed structure-activity relationship studies with bacterial LeuRS. In an in vitro assay, Kiapp values in the nanomolar range were demonstrated. Inhibitory activity differences between the compounds revealed that the polarity and size of the triazole substituents affect binding. X-ray crystallographic studies of N. gonorrhoeae LeuRS in complex with all the inhibitors highlighted the crucial interactions defining their relative enzyme inhibitory activities. We further examined their in vitro antimicrobial properties by screening against several bacterial and yeast strains. While only weak antibacterial activity against M. tuberculosis was detected, the extensive structural data which were obtained could make these LeuRS inhibitors a suitable starting point towards further antibiotic development.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leucina-tRNA Ligase/antagonistas & inibidores , Álcoois Açúcares/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Candida albicans/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Leucina-tRNA Ligase/isolamento & purificação , Leucina-tRNA Ligase/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Neisseria gonorrhoeae/enzimologia , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Álcoois Açúcares/síntese química , Álcoois Açúcares/química
4.
Molecules ; 25(20)2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-33081246

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.


Assuntos
Aminoacil-tRNA Sintetases/ultraestrutura , Benzimidazóis/química , Inibidores Enzimáticos/síntese química , Ribonucleosídeos/química , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/química , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/patogenicidade , Conformação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade
5.
Bioorg Med Chem ; 28(17): 115645, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32773091

RESUMO

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5'S, 6'R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5'R,6'S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.


Assuntos
Inibidores Enzimáticos/síntese química , Ferricromo/análogos & derivados , Serina-tRNA Ligase/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ferricromo/síntese química , Ferricromo/química , Ferricromo/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Serina-tRNA Ligase/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia
6.
Bioorg Med Chem ; 28(15): 115580, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32631562

RESUMO

Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168). Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3'- and 4'- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Isoleucina-tRNA Ligase/antagonistas & inibidores , Ácidos Sulfônicos/farmacologia , Triazóis/farmacologia , Tirosina-tRNA Ligase/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Candida/efeitos dos fármacos , Domínio Catalítico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Isoleucina-tRNA Ligase/química , Isoleucina-tRNA Ligase/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Ligação Proteica , Staphylococcus aureus/efeitos dos fármacos , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/metabolismo , Thermus thermophilus/enzimologia , Triazóis/síntese química , Triazóis/metabolismo , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo
7.
ACS Chem Biol ; 15(2): 407-415, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31869198

RESUMO

The pyrimidine-containing Trojan horse antibiotics albomycin and a recently discovered cytidine-containing microcin C analog target the class II seryl- and aspartyl-tRNA synthetases (serRS and aspRS), respectively. The active components of these compounds are competitive inhibitors that mimic the aminoacyl-adenylate intermediate. How they effectively substitute for the interactions mediated by the canonical purine group is unknown. Employing nonhydrolyzable aminoacyl-sulfamoyl nucleosides substituting the base with cytosine, uracil, and N3-methyluracil the structure-activity relationship of the natural compounds was evaluated. In vitro using E. coli serRS and aspRS, the best compounds demonstrated IC50 values in the low nanomolar range, with a clear preference for cytosine or N3-methyluracil over uracil. X-ray crystallographic structures of K. pneumoniae serRS and T. thermophilus aspRS in complex with the compounds showed the contribution of structured waters and residues in the conserved motif-2 loop in defining base preference. Utilizing the N3-methyluracil bound serRS structure, MD simulations of the fully modified albomycin base were performed to identify the interacting network that drives stable association. This analysis pointed to key interactions with a methionine in the motif-2 loop. Interestingly, this residue is mutated to a glycine in a second serRS (serRS2) found in albomycin-producing actinobacteria possessing self-immunity to this antibiotic. A comparative study demonstrated that serRS2 is poorly inhibited by the pyrimidine-containing intermediate analogs, and an equivalent mutation in E. coli serRS significantly decreased the affinity of the cytosine congener. These findings highlight the crucial role of dynamics and solvation of the motif-2 loop in modulating the binding of the natural antibiotics.


Assuntos
Antibacterianos/metabolismo , Aspartato-tRNA Ligase/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Serina-tRNA Ligase/antagonistas & inibidores , Sequência de Aminoácidos , Antibacterianos/química , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Inibidores Enzimáticos/química , Simulação de Dinâmica Molecular , Estrutura Molecular , Família Multigênica , Mutação , Ligação Proteica , Nucleosídeos de Pirimidina/química , Serina-tRNA Ligase/genética , Serina-tRNA Ligase/metabolismo , Relação Estrutura-Atividade
8.
Antibiotics (Basel) ; 8(4)2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31600972

RESUMO

Emerging antibiotic resistance in pathogenic bacteria and reduction of compounds in the existing antibiotics discovery pipeline is the most critical concern for healthcare professionals. A potential solution aims to explore new or existing targets/compounds. Inhibition of bacterial aminoacyl-tRNA synthetase (aaRSs) could be one such target for the development of antibiotics. The aaRSs are a group of enzymes that catalyze the transfer of an amino acid to their cognate tRNA and therefore play a pivotal role in translation. Thus, selective inhibition of these enzymes could be detrimental to microbes. The 5'-O-(N-(L-aminoacyl)) sulfamoyladenosines (aaSAs) are potent inhibitors of the respective aaRSs, however due to their polarity and charged nature they cannot cross the bacterial membranes. In this work, we increased the lipophilicity of these existing aaSAs in an effort to promote their penetration through the bacterial membrane. Two strategies were followed, either attaching a (permanent) alkyl moiety at the adenine ring via alkylation of the N6-position or introducing a lipophilic biodegradable prodrug moiety at the alpha-terminal amine, totaling eight new aaSA analogues. All synthesized compounds were evaluated in vitro using either a purified Escherichia coli aaRS enzyme or in presence of total cellular extract obtained from E. coli. The prodrugs showed comparable inhibitory activity to the parent aaSA analogues, indicating metabolic activation in cellular extracts, but had little effect on bacteria. During evaluation of the N6-alkylated compounds against different microbes, the N6-octyl containing congener 6b showed minimum inhibitory concentration (MIC) of 12.5 µM against Sarcina lutea while the dodecyl analogue 6c displayed MIC of 6.25 µM against Candida albicans.

9.
J Biol Chem ; 294(31): 11863-11875, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31201270

RESUMO

Only a small portion of human immunodeficiency virus type 1 (HIV-1) particles entering the host cell results in productive infection, emphasizing the importance of identifying the functional virus population. Because integration of viral DNA (vDNA) is required for productive infection, efficient vDNA detection is crucial. Here, we use click chemistry to label viruses with integrase coupled to eGFP (HIVIN-eGFP) and visualize vDNA. Because click labeling with 5-ethynyl-2'-deoxyuridine is hampered by intense background staining of the host nucleus, we opted for developing HIV-1 reverse transcriptase (RT)-specific 2'-deoxynucleoside analogs that contain a clickable triple bond. We synthesized seven propargylated 2'-deoxynucleosides and tested them for lack of cytotoxicity and viral replication inhibition, RT-specific primer extension and incorporation kinetics in vitro, and the capacity to stain HIV-1 DNA. The triphosphate of analog A5 was specifically incorporated by HIV-1 RT, but no vDNA staining was detected during infection. Analog A3 was incorporated in vitro by HIV-1 RT and human DNA polymerase γ and did enable specific HIV-1 DNA labeling. Additionally, A3 supported mitochondria-specific DNA labeling, in line with the in vitro findings. After obtaining proof-of-principle of RT-specific DNA labeling reported here, further chemical refinement is necessary to develop even more efficient HIV-1 DNA labels without background staining of the nucleus or mitochondria.


Assuntos
Química Click , Desoxiuridina/análogos & derivados , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Replicação Viral , Alcinos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA/metabolismo , Desoxiuridina/metabolismo , Desoxiuridina/toxicidade , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/genética , Humanos , Cinética , Microscopia Confocal , RNA Viral/química , RNA Viral/metabolismo
10.
Eur J Med Chem ; 174: 252-264, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31048140

RESUMO

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Šcrystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.


Assuntos
Adenosina/análogos & derivados , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/química , Sulfonamidas/química , Adenosina/síntese química , Aminoacil-tRNA Sintetases/química , Aminoacilação , Bacillus subtilis/enzimologia , Domínio Catalítico , Dickeya chrysanthemi/enzimologia , Inibidores Enzimáticos/síntese química , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Sulfolobus/enzimologia , Sulfonamidas/síntese química , Thermus thermophilus/enzimologia
11.
Eur J Med Chem ; 173: 154-166, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995568

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N3-methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated Kiapp values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleosídeos/farmacologia , Pirimidinas/farmacologia , Aminoacil-tRNA Sintetases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/síntese química , Escherichia coli/citologia , Escherichia coli/enzimologia , Estrutura Molecular , Nucleosídeos/análise , Nucleosídeos/síntese química , Pirimidinas/análise , Pirimidinas/síntese química , Relação Estrutura-Atividade
12.
Molecules ; 24(3)2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30696094

RESUMO

In vivo imaging of biological processes is an important asset of modern cell biology. Selectively reacting fluorophores herein are an important tool and click chemistry reactions take a large share in these events. 5-Ethynyl-2'-deoxyuridine (EdU) is well known for visualizing DNA replication, but does not show any selectivity for incorporation into DNA. Striving for specific visualization of virus replication, in particular HIV replication, a series of propargylated purine deoxynucleosides were prepared aiming for selective incorporation by HIV reverse transcriptase (RT). We here report on the synthesis and preliminary biological effects (cellular toxicity, HIV inhibitory effects, and feasibility of the click reaction) of these nucleoside analogues.


Assuntos
Corantes Fluorescentes , Imagem Óptica , Nucleosídeos de Purina , Linhagem Celular , Sobrevivência Celular , Química Click , Corantes Fluorescentes/química , Expressão Gênica , Genes Reporter , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Estrutura Molecular , Imagem Óptica/métodos , Nucleosídeos de Purina/química , Replicação Viral/efeitos dos fármacos
13.
Eur J Med Chem ; 148: 384-396, 2018 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-29477072

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that precisely attach an amino acid to its cognate tRNA. This process, which is essential for protein translation, is considered a viable target for the development of novel antimicrobial agents, provided species selective inhibitors can be identified. Aminoacyl-sulfamoyl adenosines (aaSAs) are potent orthologue specific aaRS inhibitors that demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work on substitution of the base moiety, we evaluated the effect of the N3-position of the adenine by synthesizing the corresponding 3-deazaadenosine analogues (aaS3DAs). A typical organism has 20 different aaRS, which can be split into two distinct structural classes. We therefore coupled six different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the presence of a conserved water molecule which could have importance for base recognition within class II enzymes, a property that can be explored in future drug design efforts.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Infecciosos/química , Tubercidina/química , Aminoácidos/química , Desenho de Fármacos , Proteínas de Escherichia coli , Ácidos Sulfônicos/química , Tubercidina/farmacologia
14.
Mol Pharm ; 14(5): 1726-1741, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28363028

RESUMO

We recently found that indomethacin (IMC) can effectively act as a powerful crystallization inhibitor for polyethylene glycol 6000 (PEG) despite the fact that the absence of interactions between the drug and the carrier in the solid state was reported in the literature. However, in the present study, we investigate the possibility of drug-carrier interactions in the liquid state to explain the polymer crystallization inhibition effect of IMC. We also aim to discover other potential PEG crystallization inhibitors. Drug-carrier interactions in both liquid and solid state are characterized by variable temperature Fourier transform infrared spectroscopy (FTIR) and cross-polarization magic angle spinning 13C nuclear magnetic resonance spectroscopy (CP/MAS NMR). In the liquid state, FTIR data show evidence of the breaking of hydrogen bonding between IMC molecules to form interactions of the IMC monomer with PEG. The drug-carrier interactions are disrupted upon storage and polymer crystallization, resulting in segregation of IMC from PEG crystals that can be observed under polarized light microscopy. This process is further confirmed by 13C NMR since in the liquid state, when the IMC/PEG monomer units ratio is below 2:1, IMC signals are undetectable because of the loss of cross-polarization efficiency in the mobile IMC molecules upon attachment to PEG chains via hydrogen bonding. This suggests that each ether oxygen of the PEG unit can form hydrogen bonds with two IMC molecules. The NMR spectrum of IMC shows no change in solid dispersions with PEG upon storage, indicating the absence of interactions in the solid state, hence confirming previous studies. The drug-carrier interactions in the liquid state elucidate the crystallization inhibition effect of IMC on PEG as well as other semicrystalline polymers such as poloxamer and Gelucire. However, hydrogen bonding is a necessary but apparently not a sufficient condition for the polymer crystallization inhibition. Screening of crystallization inhibitors of semicrystalline polymers discovers numerous candidates that exhibit the same behavior as IMC, demonstrating a general pattern of polymer crystallization inhibition rather than a particular case. Furthermore, the crystallization inhibition effect of drugs on PEG is independent of the carrier molecular weight. These mechanistic findings on the formation and disruption of hydrogen bonds in semicrystalline dispersions can be extended to amorphous dispersions and are of significant importance for preparation of solid dispersions with consistent and reproducible physicochemical properties.


Assuntos
Indometacina/química , Varredura Diferencial de Calorimetria , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Polietilenoglicóis/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
15.
Eur J Med Chem ; 126: 101-109, 2017 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-27750144

RESUMO

Previous efforts led to dicarboxamide derivatives like 1.3, comprising either an imidazole, pyrazine or fenyl ring as the central scaffold, with many congeners displaying strong inhibitory effects against dengue virus (DENV) in cell-based assays. Following up on some literature reports, the rationale was borne out to preserve the pending groups, now attached to either a 2,6-diaminopurine or 2,4-diaminoquinazoline scaffold. Synthetic efforts turned out less straightforward than expected, but yielded some new derivatives with low micromolar anti-DENV activity, albeit not devoid of cellular toxicity. The purine 14 proved the most potent compound for this series with an EC50 of 1.9 µM and a selectivity index of 58, while the quinazoline 18a displayed an EC50 of 2.6 µM with SI of only 2.


Assuntos
2-Aminopurina/análogos & derivados , Antivirais/química , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Quinazolinas/química , Quinazolinas/farmacologia , 2-Aminopurina/química , 2-Aminopurina/metabolismo , 2-Aminopurina/farmacologia , Antivirais/metabolismo , Vírus da Dengue/enzimologia , Desenho de Fármacos , Simulação de Acoplamento Molecular , Conformação Proteica , Quinazolinas/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
16.
J Am Chem Soc ; 138(48): 15690-15698, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27934031

RESUMO

Microcin C and related antibiotics are Trojan-horse peptide-adenylates. The peptide part is responsible for facilitated transport inside the sensitive cell, where it gets processed to release a toxic warhead-a nonhydrolyzable aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. Adenylation of peptide precursors is carried out by MccB THIF-type NAD/FAD adenylyltransferases. Here, we describe a novel microcin C-like compound from Bacillus amyloliquefaciens. The B. amyloliquefaciens MccB demonstrates an unprecedented ability to attach a terminal cytidine monophosphate to cognate precursor peptide in cellular and cell free systems. The cytosine moiety undergoes an additional modification-carboxymethylation-that is carried out by the C-terminal domain of MccB and the MccS enzyme that produces carboxy-SAM, which serves as a donor of the carboxymethyl group. We show that microcin C-like compounds carrying terminal cytosines are biologically active and target aspartyl-tRNA synthetase, and that the carboxymethyl group prevents resistance that can occur due to modification of the warhead. The results expand the repertoire of known enzymatic modifications of peptides that can be used to obtain new biological activities while avoiding or limiting bacterial resistance.


Assuntos
Antibacterianos/farmacologia , Aspartato-tRNA Ligase/antagonistas & inibidores , Bacillus amyloliquefaciens/química , Bacteriocinas/farmacologia , Inibidores Enzimáticos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Biologia Computacional , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Estrutura Molecular
17.
Eur J Med Chem ; 121: 158-168, 2016 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-27240271

RESUMO

High-throughput screening of a subset of the CD3 chemical library (Centre for Drug Design and Discovery; KU Leuven) provided us with a lead compound 1, displaying low micromolar potency against dengue virus and yellow fever virus. Within a project aimed at discovering new inhibitors of flaviviruses, substitution of its central imidazole ring led to synthesis of variably substituted pyrazine dicarboxylamides and phthalic diamides, which were evaluated in cell-based assays for cytotoxicity and antiviral activity against the dengue virus (DENV) and yellow fever virus (YFV). Fourteen compounds inhibited DENV replication (EC50 ranging between 0.5 and 3.4 µM), with compounds 6b and 6d being the most potent inhibitors (EC50 0.5 µM) with selectivity indices (SI) > 235. Compound 7a likewise exhibited anti-DENV activity with an EC50 of 0.5 µM and an SI of >235. In addition, good antiviral activity of seven compounds in the series was also noted against the YFV with EC50 values ranging between 0.4 and 3.3 µM, with compound 6n being the most potent for this series with an EC50 0.4 µM and a selectivity index of >34. Finally, reversal of one of the central amide bonds as in series 13 proved deleterious to the inhibitory activity.


Assuntos
Vírus da Dengue/efeitos dos fármacos , Diamida/farmacologia , Compostos Heterocíclicos/farmacologia , Vírus da Febre Amarela/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacologia , Chlorocebus aethiops , Diamida/química , Descoberta de Drogas/métodos , Compostos Heterocíclicos/química , Relação Estrutura-Atividade , Células Vero , Replicação Viral/efeitos dos fármacos
18.
Org Biomol Chem ; 13(39): 10041-9, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26293202

RESUMO

The synthesis and a preliminary evaluation of the pairing properties of ribo-cyclohexanyl nucleic acids (r-CNA) is herein reported. Incorporation of a single r-CNA nucleotide into natural duplexes did not enhance their stability, while a very high pairing selectivity for RNA was found. As deduced by comparative analysis of Tm and NMR data, a relationship between pairing selectivity and conformational preferences of the "sugar" moiety of r-CNA (and more generally of six-membered nucleic acids) was suggested.


Assuntos
Pareamento de Bases , Oligonucleotídeos/química , RNA/química , Ribonucleosídeos/química , Ribose/análogos & derivados , Sequência de Bases , Configuração de Carboidratos , Conformação de Ácido Nucleico , Estabilidade de RNA
19.
J Org Chem ; 80(10): 5014-22, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25853790

RESUMO

The synthesis and a preliminary investigation of the base pairing properties of (6' → 4')-linked 1',5'-anhydro-L-ribo-hexitol nucleic acids (α-L-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the "all equatorial" pyranyl sugar backbone, α-L-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-L-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids.


Assuntos
Adenina/síntese química , Ácidos Nucleicos/síntese química , Oligonucleotídeos/síntese química , Álcoois Açúcares/síntese química , Adenina/química , Pareamento de Bases , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Oligonucleotídeos/química , RNA/química , Estereoisomerismo , Álcoois Açúcares/química
20.
Curr Med Chem ; 22(18): 2140-58, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25787965

RESUMO

With the alarming resistance to currently used antibiotics, there is a serious worldwide threat to public health. Therefore, there is an urgent need to search for new antibiotics or new cellular targets which are essential for survival of the pathogens. However, during the past 50 years, only two new classes of antibiotics (oxazolidinone and lipopeptides) have reached the clinic. This suggests that the success rate in discovering new/novel antibiotics using conventional approaches is limited and that we must reconsider our antibiotic discovery approaches. While many new strategies are being pursued lately, this review primarily focuses only on a few of these novel/new approaches for antibiotic discovery. These include structure-based drug design (SBDD), the genomic approach, anti-virulence strategy, targeting nonmultiplying bacteria and the use of bacteriophages. In general, recent advancements in nuclear magnetic resonance, Xcrystallography, and genomic evolution have significant impact on antibacterial drug research. This review therefore aims to discuss recent strategies in searching new antibacterial agents making use of these technical novelties, their advantages, disadvantages and limitations.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Descoberta de Drogas , Antibacterianos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular
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