RESUMO
The field of synthetic nucleic acids with novel backbone structures [xenobiotic nucleic acids (XNAs)] has flourished due to the increased importance of XNA antisense oligonucleotides and aptamers in medicine, as well as the development of XNA processing enzymes and new XNA genetic materials. Molecular modeling on XNA structures can accelerate rational design in the field of XNAs as it contributes in understanding and predicting how changes in the sugar-phosphate backbone impact on the complementation properties of the nucleic acids. To support the development of novel XNA polymers, we present a first-in-class open-source program (Ducque) to build duplexes of nucleic acid analogs with customizable chemistry. A detailed procedure is described to extend the Ducque library with new user-defined XNA fragments using quantum mechanics (QM) and to generate QM-based force field parameters for molecular dynamics simulations within standard packages such as AMBER. The tool was used within a molecular modeling workflow to accurately reproduce a selection of experimental structures for nucleic acid duplexes with ribose-based as well as non-ribose-based nucleosides. Additionally, it was challenged to build duplexes of morpholino nucleic acids bound to complementary RNA sequences.
Assuntos
Simulação de Dinâmica Molecular , Morfolinos , Ácidos Nucleicos , RNA , Software , Morfolinos/química , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Oligonucleotídeos/química , RNA/química , Software/normasRESUMO
In this study, we investigated how the replacement of the tetrahydrothiophene ring of biotin with either an oxolane or (methyl)pyrrolidine moiety may affect its molecular interactions, in an effort to identify alternative affinity ligands suitable for in vitro and in vivo applications in synthetic biology. Initial molecular dynamics (MD) simulations suggested the potential formation of a hydrogen bond between either the oxygen or nitrogen atom of the envisaged tetrahydroheteryl analogues and the Thr90 residue of streptavidin, mirroring the sulfur-centered hydrogen bond detected by the crystallographic analysis of the biotin-streptavidin interaction. Therefore, oxy-, aza-, and N-methylazabiotin were readily synthesized starting from chiral five- or six-carbon sugar precursors. Based on fluorescence-based titration experiments using the corresponding fluorescein conjugates, oxybiotin showed a binding behavior similar to biotin with streptavidin, while both amino analogues displayed lower binding capacities. Notably, azabiotin exhibited a pH-dependent interaction profile, demonstrating enhanced binding under acidic conditions but weaker binding under basic pH, which could be exploited for various purposes.
Assuntos
Biotina , Estreptavidina , Enxofre , Biotina/química , Estreptavidina/química , Estrutura Molecular , Enxofre/química , Sítios de Ligação , Simulação de Dinâmica Molecular , Ligação Proteica , Ligação de HidrogênioRESUMO
Disubstituted isothiazolo[4,3-b]pyridines are known inhibitors of cyclin G-associated kinase. Since 3-substituted-7-aryl-isothiazolo[4,3-b]pyridines remain elusive, a strategy was established to prepare this chemotype, starting from 2,4-dichloro-3-nitropyridine. Selective C-4 arylation using ligand-free Suzuki-Miyaura coupling and palladium-catalyzed aminocarbonylation functioned as key steps in the synthesis. The 3-N-morpholinyl-7-(3,4-dimethoxyphenyl)-isothiazolo[4,3-b]pyridine was completely devoid of GAK affinity, in contrast to its 3,5- and 3,6-disubstituted congeners. Molecular modeling was applied to rationalize its inactivity as a GAK ligand.
Assuntos
Paládio , Piridinas , Piridinas/farmacologia , Modelos Moleculares , Ligantes , Ciclina G , CatáliseRESUMO
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/químicaRESUMO
Previously, we reported the discovery of 3,6-disubstituted isothiazolo[4,3-b]pyridines as potent and selective cyclin G-associated kinase (GAK) inhibitors with promising antiviral activity. In this manuscript, the structure-activity relationship study was expanded to synthesis of isothiazolo[4,3-b]pyridines with modifications of the pyridine moiety. This effort led to the discovery of an isothiazolo[4,3-b]pyridine derivative with a 3,4-dimethoxyphenyl residue at position 5 that displayed low nanomolar GAK binding affinity and antiviral activity against dengue virus.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Tiazóis/farmacologia , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a "hot spot" for the inhibition of pestivirus replication.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/tratamento farmacológico , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Pestivirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/patogenicidade , Farmacorresistência Viral/genética , Mutação/efeitos dos fármacos , Pestivirus/patogenicidade , Quinolinas/farmacologiaRESUMO
A synthetic orthogonal polymer embracing a chiral acyclic-phosphonate backbone [(S)-ZNA] is presented that uniquely adds to the emerging family of xenobiotic nucleic acids (XNAs). (S)-ZNA consists of reiterating six-atom structural units and can be accessed in few synthetic steps from readily available phophonomethylglycerol nucleoside (PMGN) precursors. Comparative thermal stability experiments conducted on homo- and heteroduplexes made of (S)-ZNA are described that evince its high self-hybridization efficiency in contrast to poor binding of natural complements. Although preliminary and not conclusive, circular dichroism data and dynamic modeling computations provide support to a left-handed geometry of double-stranded (S)-ZNA. Nonetheless, PMGN diphosphate monomers were recognized as substrates by Escherichia coli (E. coli) polymerase I as well as being imported into E. coli cells equipped with an algal nucleotide transporter. A further investigation into the in vivo propagation of (S)-ZNA culminated with the demonstration of the first synthetic nucleic acid with an acyclic backbone that can be transliterated to DNA by the E. coli cellular machinery.
Assuntos
Escherichia coli/genética , Ácidos Nucleicos/química , Organofosfonatos/química , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ácidos Nucleicos/genética , Oligonucleotídeos/química , Oligonucleotídeos/genéticaRESUMO
A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable MtbTMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3. Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC50=35 µM). This finding opens avenues for future modifications.
Assuntos
Amidas/farmacologia , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Timina/farmacologia , Amidas/síntese química , Amidas/química , Antituberculosos/síntese química , Antituberculosos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Núcleosídeo-Fosfato Quinase/metabolismo , Relação Estrutura-Atividade , Timina/síntese química , Timina/químicaRESUMO
Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.IMPORTANCE In this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.
Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/genética , Lisina/metabolismo , Pirazinas/farmacologia , RNA Polimerase Dependente de RNA/química , Amidas/administração & dosagem , Motivos de Aminoácidos , Animais , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/genética , Chlorocebus aethiops , Farmacorresistência Viral/genética , Enterovirus Humano B/enzimologia , Japão , Lisina/genética , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Mutagênese , Mutação , Pirazinas/administração & dosagem , RNA Polimerase Dependente de RNA/genética , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The paradigm of homogenous-sugar-backbone of RNA and DNA has reliably guided the construction of many functional and useful xeno nucleic acid (XNA) systems to date. Deviations from this monotonous and canonical design, in many cases, results in oligonucleotide systems that lack base pairing with themselves, or with RNA or DNA. Here we show that nucleotides of two such compromised XNA systems can be combined with RNA and DNA in specific patterns to produce chimeric-backbone oligonucleotides, which in certain cases demonstrate base pairing properties comparable to-or stronger than-canonical systems, while also altering the conventional Watson-Crick pairing behavior. The unorthodox pairing properties generated from these chimeric sugar-backbone oligonucleotides suggest a counterintuitive approach of creating modules consisting of non-base pairing XNAs with RNA/DNA in a set pattern. This strategy has the potential to increase the diversity of unconventional nucleic acids leading to orthogonal backbone-sequence-controlled informational systems.
Assuntos
DNA/química , Nucleotídeos/química , RNA/química , Pareamento de Bases , Dimerização , Ligação de Hidrogênio , Modelos Moleculares , Oligonucleotídeos/química , TermodinâmicaRESUMO
We investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided.
Assuntos
Antivirais/farmacologia , Enterovirus Humano D/efeitos dos fármacos , Infecções por Enterovirus/virologia , Farmacorresistência Viral , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oxidiazóis/farmacologia , Oxazóis , Receptores de Droga/química , Receptores de Droga/efeitos dos fármacos , Infecções Respiratórias/virologia , Proteínas Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
The synthesis and a preliminary investigation of the base pairing properties of (6' â 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/químicaRESUMO
In further study of our series of six-membered ring-containing nucleic acids, different 1',3'-di-O-methyl altropyranoside nucleoside analogs (DMANA) were synthesized comprising all four base moieties, adenine, cytosine, uracil and guanine. Following assembly into oligonucleotides (ONs), their affinity for natural oligonucleotides was evaluated by thermal denaturation of the respective duplexes. Data were compared with results obtained previously for both anhydrohexitol (HNAs) and 3'-O-methylated altrohexitol modified ONs (MANAs). We hereby demonstrate that ONs modified with DMANA monomers, unlike some of our previously described analogues with constrained 6-membered hexitol rings, did not improve thermodynamic stability of dsRNA complexes, most probably in view of an energetic penalty when forced in the required 1C4 pairing conformation. Overall, a single incorporation was more or less tolerated or even positive for the adenine congener, but incorporation of a second modification afforded a slight destabilization (except for A), while a fully modified sequence displayed a thermal stability of -0.3 °C per modification. The selectivity of pairing remained very high, and the new modification upon incorporation into a DNA strand, strongly destabilized the corresponding DNA duplexes. Unfortunately, this new modification does not bring any advantage to be further evaluated for antisense or siRNA applications.
Assuntos
Hibridização de Ácido Nucleico/genética , Ácidos Nucleicos/química , Adenina/química , Citosina/química , DNA/química , DNA/genética , Guanina/química , Desnaturação de Ácido Nucleico/genética , Oligonucleotídeos/química , RNA Interferente Pequeno/genética , Álcoois Açúcares/química , Termodinâmica , Uracila/químicaRESUMO
Antivirals against enterovirus 71 (EV71) are urgently needed. We demonstrate that the novel enteroviral protease inhibitor (PI) SG85 and capsid binder (CB) vapendavir efficiently inhibit the in vitro replication of 21 EV71 strains/isolates that are representative of the different genogroups A, B, and C. The PI rupintrivir, the CB pirodavir, and the host-targeting compound enviroxime, which were included as reference compounds, also inhibited the replication of all isolates. Remarkably, the CB compound pleconaril was devoid of any anti-EV71 activity. An in silico docking study revealed that pleconaril-unlike vapendavir and pirodavir-lacks essential binding interactions with the viral capsid. Vapendavir and SG85 (or analogues) should be further explored for the treatment of EV71 infections. The data presented here may serve as a reference when developing yet-novel inhibitors.
Assuntos
Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Replicação Viral/efeitos dos fármacos , Benzimidazóis/farmacologia , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Farmacorresistência Viral/genética , Enterovirus Humano A/classificação , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Isoxazóis/farmacologia , Simulação de Acoplamento Molecular , Oximas , Fenilalanina/análogos & derivados , Piperidinas/farmacologia , Ligação Proteica , Piridazinas/farmacologia , Pirrolidinonas/farmacologia , Sulfonamidas , Valina/análogos & derivadosRESUMO
OBJECTIVES: To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). METHODS: The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. RESULTS: LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2â±â1 µM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. CONCLUSIONS: LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication.
Assuntos
Antivirais/farmacologia , Nitrilas/farmacologia , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Sítios de Ligação , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Genótipo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Molecular , Mutação , Nitrilas/química , Ligação Proteica , RNA Viral , Rhinovirus/genética , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
OBJECTIVES: T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus. METHODS: The anti-CHIKV effect of favipiravir was examined in cell culture and in a mouse model of lethal infection. A five-step protocol was used to select for CHIKV variants with reduced susceptibility to favipiravir. The resistant phenotype was confirmed in cell culture and the whole genome was sequenced. The identified mutations were reverse-engineered into an infectious clone to confirm their impact on the antiviral efficacy of favipiravir. RESULTS: Favipiravir inhibits the replication of laboratory strains and clinical isolates of CHIKV, as well as of a panel of other alphaviruses. Several favipiravir-resistant CHIKV variants were independently selected and all of them in particular acquired the unique K291R mutation in the RNA-dependent RNA polymerase (RdRp). Reverse-engineering of this K291R mutation into an infectious clone of CHIKV confirmed the link between the mutant genotype and the resistant phenotype. Interestingly, this particular lysine is also highly conserved in the RdRp of positive-stranded RNA viruses in general. CONCLUSIONS: This study provides an important insight into the precise molecular mechanism by which favipiravir exerts its antiviral activity against (alpha)viruses, which may be of help in designing other potent broad-spectrum antivirals.
Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/genética , Farmacorresistência Viral/genética , Mutação , Pirazinas/farmacologia , Proteínas não Estruturais Virais/genética , Amidas/química , Animais , Antivirais/química , Linhagem Celular , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/virologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Camundongos , Testes de Sensibilidade Microbiana , Fenótipo , Pirazinas/química , Reprodutibilidade dos Testes , Replicação Viral/efeitos dos fármacosRESUMO
Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of the corresponding prokaryotic and eukaryotic tRNA synthetases in vitro. Inspired by the aryl-tetrazole containing compounds of Cubist Pharmaceuticals and the modified base as found in the natural antibiotic albomycin, the selectivity issue of the sulfamoylated adenosines prompted us to investigate the pharmacophoric importance of the adenine base. We therefore synthesized and evaluated several isoleucyl-sulfamoyl nucleoside analogues with either uracil, cytosine, hypoxanthine, guanine, 1,3-dideaza-adenine (benzimidazole) or 4-nitro-benzimidazole as the heterocyclic base. Based on the structure and antibacterial activity of microcin C, we also prepared their hexapeptidyl conjugates in an effort to improve their uptake potential. We further compared their antibacterial activity with the parent isoleucyl-sulfamoyl adenosine (Ile-SA), both in in vitro and in cellular assays. Surprisingly, the strongest in vitro inhibition was found for the uracil containing analogue 16f. Unfortunately, only very weak growth inhibitory properties were found as of low uptake. The results are discussed in the light of previous literature findings.
Assuntos
Adenosina/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Adenosina/análogos & derivados , Adenosina/química , Antibacterianos/síntese química , Relação Dose-Resposta a Droga , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Antibiotic resistance has increased over the past two decades. New approaches for the discovery of novel antibacterials are required and innovative strategies will be necessary to identify novel and effective candidates. Related to this problem, the exploration of bacterial targets that remain unexploited by the current antibiotics in clinical use is required. One of such targets is the ß-ketoacyl-acyl carrier protein synthase III (FabH). Here, we report a ligand-based modeling methodology for the virtual-screening of large collections of chemical compounds in the search of potential FabH inhibitors. QSAR models are developed for a diverse dataset of 296 FabH inhibitors using an in-house modeling framework. All models showed high fitting, robustness, and generalization capabilities. We further investigated the performance of the developed models in a virtual screening scenario. To carry out this investigation, we implemented a desirability-based algorithm for decoys selection that was shown effective in the selection of high quality decoys sets. Once the QSAR models were validated in the context of a virtual screening experiment their limitations arise. For this reason, we explored the potential of ensemble modeling to overcome the limitations associated to the use of single classifiers. Through a detailed evaluation of the virtual screening performance of ensemble models it was evidenced, for the first time to our knowledge, the benefits of this approach in a virtual screening scenario. From all the obtained results, we could arrive to a significant main conclusion: at least for FabH inhibitors, virtual screening performance is not guaranteed by predictive QSAR models.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Relação Quantitativa Estrutura-Atividade , Interface Usuário-Computador , Escherichia coli/enzimologia , Ligantes , Modelos MolecularesRESUMO
Nitrogen-containing steroids are known as prostate cancer therapeutics. In this work, a series of pregnane derivatives bearing an imidazolium moiety were synthesized using Δ16-20-ketones as starting material. An improved approach for the construction of the 20-keto-21-heterocycle-substituted fragment involved the rearrangement of 16,17-epoxides with HCl, followed by reaction of the formed α-chloroketone with 1-substituted imidazoles. Binding affinity analysis of the imidazolium steroids and their synthetic intermediates to human CYP17A1 showed only type I (substrate-like) interactions. The strongest affinity was observed for 16α,17α-epoxy-5α-pregnan-20-on-3ß-ol (Kd = 0.66 ± 0.05 µM). The steroid derivatives have been evaluated for antitumor activity against a range of prostate cancer cells as well as against various other solid tumor and hematologic cancer cell lines. All 21-imidazolium salts were active against the hormone-dependent prostate cancer line LNCaP. The most pronounced cytotoxicity in solid tumor and hematologic cancer cell lines was observed for intermediate product, 21-chloro-5α-pregn-16-en-20-on-3ß-ol. Among the imidazolium salts, the derivatives with a single bond were more cytotoxic than their unsaturated congeners.