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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.
Sci Rep ; 11(1): 5433, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686143

RESUMO

The lack of coronavirus-specific antiviral drugs has instigated multiple drug repurposing studies to redirect previously approved medicines for the treatment of SARS-CoV-2, the coronavirus behind the ongoing COVID-19 pandemic. A recent, large-scale, retrospective clinical study showed that famotidine, when administered at a high dose to hospitalized COVID-19 patients, reduced the rates of intubation and mortality. A separate, patient-reported study associated famotidine use with improvements in mild to moderate symptoms such as cough and shortness of breath. While a prospective, multi-center clinical study is ongoing, two parallel in silico studies have proposed one of the two SARS-CoV-2 proteases, 3CLpro or PLpro, as potential molecular targets of famotidine activity; however, this remains to be experimentally validated. In this report, we systematically analyzed the effect of famotidine on viral proteases and virus replication. Leveraging a series of biophysical and enzymatic assays, we show that famotidine neither binds with nor inhibits the functions of 3CLpro and PLpro. Similarly, no direct antiviral activity of famotidine was observed at concentrations of up to 200 µM, when tested against SARS-CoV-2 in two different cell lines, including a human cell line originating from lungs, a primary target of COVID-19. These results rule out famotidine as a direct-acting inhibitor of SARS-CoV-2 replication and warrant further investigation of its molecular mechanism of action in the context of COVID-19.


Assuntos
Famotidina/farmacologia , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/enzimologia , Replicação Viral/efeitos dos fármacos , Células A549 , Animais , COVID-19/virologia , Chlorocebus aethiops , Humanos , SARS-CoV-2/efeitos dos fármacos , Células Vero
3.
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
4.
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
5.
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
6.
Int J Mol Sci ; 21(16)2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824134

RESUMO

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being critically involved in fibrinolysis and wound healing, emerging evidence indicates that PAI-1 plays an important role in many diseases, including cardiovascular disease, tissue fibrosis, and cancer. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1 related pathologies. Despite ongoing efforts no PAI-1 inhibitors were approved to date for therapeutic use in humans. A better understanding of the molecular mechanisms of PAI-1 inhibition is therefore necessary to guide the rational design of PAI-1 modulators. Here, we present a 1.9 Å crystal structure of PAI-1 in complex with an inhibitory nanobody VHH-s-a93 (Nb93). Structural analysis in combination with biochemical characterization reveals that Nb93 directly interferes with PAI-1/PA complex formation and stabilizes the active conformation of the PAI-1 molecule.


Assuntos
Simulação de Acoplamento Molecular , Inibidor 1 de Ativador de Plasminogênio/química , Anticorpos de Domínio Único/química , Sítios de Ligação , Humanos , Inibidor 1 de Ativador de Plasminogênio/imunologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/imunologia
7.
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
8.
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
9.
J Thromb Haemost ; 18(3): 681-692, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31858714

RESUMO

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes (e.g., cardiovascular disease, tissue fibrosis) as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors. OBJECTIVE: To elucidate the molecular mechanism of nanobody-induced PAI-1 inhibition. METHODS AND RESULTS: Here we present the first crystal structures of PAI-1 in complex with two neutralizing nanobodies (Nbs). These structures, together with biochemical and biophysical characterization, reveal that Nb VHH-2g-42 (Nb42) interferes with the initial PAI-1/PA complex formation, whereas VHH-2w-64 (Nb64) redirects the PAI-1/PA interaction to PAI-1 deactivation and regeneration of active PA. Furthermore, whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo. CONCLUSIONS: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions.


Assuntos
Inibidor 1 de Ativador de Plasminogênio , Anticorpos de Domínio Único , Humanos , Ativadores de Plasminogênio , Ativador de Plasminogênio Tecidual , Ativador de Plasminogênio Tipo Uroquinase , Vitronectina
10.
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
12.
Nucleic Acids Res ; 47(13): 7130-7142, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31334814

RESUMO

Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.


Assuntos
DNA Ligases/metabolismo , Proteínas Virais/metabolismo , Simulação por Computador , DNA Ligases/química , Vírus de DNA/enzimologia , DNA Viral/metabolismo , Desoxirribonuclease BamHI/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Moldes Genéticos , Proteínas Virais/química
13.
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
14.
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
15.
J Struct Biol X ; 3: 100011, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32647816

RESUMO

The MJD family of human deubiquitinating enzymes contains four members: Ataxin-3, the ataxin-3-like protein (AT3L), Josephin-1, and Josephin-2. All share a conserved catalytic unit known as the Josephin domain. Ataxin-3 and AT3L also contain extensive regulatory regions that modulate their functions, whereas Josephins-1 and -2 are substantially smaller, containing only the Josephin domain. To gain insight into how these minimal Josephins differ from their larger relatives, we determined the 2.3 ŠX-ray crystal structure of human Josephin-2 and probed the enzyme's substrate specificity. Several large disordered loops are seen in the structure, suggesting a highly dynamic enzyme. Josephin-2 lacks several allosteric sites found in ataxin-3, but its structure suggests potential regulation via ubiquitination of a loop adjoining the active site. The enzyme preferentially recognizes substrates containing K11, K48, and K63 linkages, pointing toward a possible role in maintenance of protein quality control.

16.
Int J Mol Sci ; 19(7)2018 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-30036999

RESUMO

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.


Assuntos
Motivos de Aminoácidos/fisiologia , Arginina/química , Cristalinas/química , Proteínas de Choque Térmico HSP20/química , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/metabolismo , Proteínas de Choque Térmico/química , Proteínas Serina-Treonina Quinases/química , Cadeia B de alfa-Cristalina/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Arginina/genética , Cromatografia em Gel , Cristalinas/genética , Cristalinas/metabolismo , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo
17.
J Biol Chem ; 293(28): 11154-11165, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29794027

RESUMO

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in Paenibacillus alvei, which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.


Assuntos
Proteínas de Bactérias/metabolismo , Dipeptídeos/metabolismo , Endopeptidases/metabolismo , Paenibacillus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cristalografia por Raios X , Dipeptídeos/química , Endopeptidases/química , Modelos Moleculares , Paenibacillus/crescimento & desenvolvimento , Conformação Proteica , Homologia de Sequência , Especificidade por Substrato
18.
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
19.
Sci Rep ; 8(1): 688, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330367

RESUMO

Congenital mutations in human small heat shock protein HSPB1 (HSP27) have been linked to Charcot-Marie-Tooth disease, a commonly occurring peripheral neuropathy. Understanding the molecular mechanism of such mutations is indispensable towards developing future therapies for this currently incurable disorder. Here we describe the physico-chemical properties of the autosomal dominant HSPB1 mutants R127W, S135F and R136W. Despite having a nominal effect on thermal stability, the three mutations induce dramatic changes to quaternary structure. At high concentrations or under crowding conditions, the mutants form assemblies that are approximately two times larger than those formed by the wild-type protein. At low concentrations, the mutants have a higher propensity to dissociate into small oligomers, while the dissociation of R127W and R135F mutants is enhanced by MAPKAP kinase-2 mediated phosphorylation. Specific differences are observed in the ability to form hetero-oligomers with the homologue HSPB6 (HSP20). For wild-type HSPB1 this only occurs at or above physiological temperature, whereas the R127W and S135F mutants form hetero-oligomers with HSPB6 at 4 °C, and the R136W mutant fails to form hetero-oligomers. Combined, the results suggest that the disease-related mutations of HSPB1 modify its self-assembly and interaction with partner proteins thus affecting normal functioning of HSPB1 in the cell.


Assuntos
Doença de Charcot-Marie-Tooth/patologia , Proteínas de Choque Térmico HSP27/metabolismo , Sequência de Aminoácidos , Doença de Charcot-Marie-Tooth/metabolismo , Cromatografia em Gel , Difusão Dinâmica da Luz , Proteínas de Choque Térmico HSP20/química , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares , Fosforilação , Polimorfismo de Nucleotídeo Único , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Temperatura , Difração de Raios X , alfa-Cristalinas/química
20.
J Biol Chem ; 292(24): 9944-9957, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28487364

RESUMO

Small heat-shock proteins (sHSPs) are a conserved group of molecular chaperones with important roles in cellular proteostasis. Although sHSPs are characterized by their small monomeric weight, they typically assemble into large polydisperse oligomers that vary in both size and shape but are principally composed of dimeric building blocks. These assemblies can include different sHSP orthologues, creating additional complexity that may affect chaperone activity. However, the structural and functional properties of such hetero-oligomers are poorly understood. We became interested in hetero-oligomer formation between human heat-shock protein family B (small) member 1 (HSPB1) and HSPB6, which are both highly expressed in skeletal muscle. When mixed in vitro, these two sHSPs form a polydisperse oligomer array composed solely of heterodimers, suggesting preferential association that is determined at the monomer level. Previously, we have shown that the sHSP N-terminal domains (NTDs), which have a high degree of intrinsic disorder, are essential for the biased formation. Here we employed iterative deletion mapping to elucidate how the NTD of HSPB6 influences its preferential association with HSPB1 and show that this region has multiple roles in this process. First, the highly conserved motif RLFDQXFG is necessary for subunit exchange among oligomers. Second, a site ∼20 residues downstream of this motif determines the size of the resultant hetero-oligomers. Third, a region unique to HSPB6 dictates the preferential formation of heterodimers. In conclusion, the disordered NTD of HSPB6 helps regulate the size and stability of hetero-oligomeric complexes, indicating that terminal sHSP regions define the assembly properties of these proteins.


Assuntos
Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Modelos Moleculares , Motivos de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Reagentes para Ligações Cruzadas/farmacologia , Dimerização , Deleção de Genes , Proteínas de Choque Térmico HSP20/química , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Reagentes de Sulfidrila/farmacologia
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