RESUMO
The synthesis of the first dimeric inhibitor of E. coli dihydrodipicolinate synthase (DHDPS) is reported herein. Inspired by 2,4-thiazolidinedione based ligands previously shown to inhibit DHDPS, a series of dimeric inhibitors were designed and synthesised, incorporating various alkyl chain bridges between two 2,4-thiazolidinedione moieties. Aiming to exploit the multimeric nature of this enzyme and enhance potency, a dimeric compound with a single methylene bridge achieved the desired outcome with low micromolar inhibition of E. coli DHDPS observed. This work highlights the continued importance of investigation into DHDPS as an antibacterial target. Furthermore, we demonstrate the design of dimeric ligands can provide a promising strategy to improve potency in the search for novel bioactive compounds.
RESUMO
Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.
Assuntos
Dinaminas , Quinazolinonas , Humanos , Dinaminas/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial , Quinazolinonas/farmacologiaRESUMO
Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.
Assuntos
Herbicidas , Herbicidas/farmacologia , Lisina , Plantas Daninhas , Controle de Plantas DaninhasRESUMO
Chronic activation of beta-adrenergic receptors by the sympathetic nervous system results in the apoptosis of cardiomyocytes. Due to the inability of cardiomyocytes to regenerate, this can result in heart failure. Upregulation of the pro-apoptotic protein Bim has been implicated as the cause of cardiomyocyte apoptosis. Beta blockers are the frontline drug used to negate this apoptotic pathway, as no direct inhibitors of Bim expression currently exist. Unfortunately, treatment of heart failure using beta blockers is not optimal. Therefore, direct inhibition of Bim expression is an attractive strategy to provide protection against stress-induced apoptosis of cardiomyocytes. Herein we explore a class of N-benzylsulfonyl-2-phenylazepanes to obtain anti-apoptotic compounds capable of reducing Bim expression levels to 7% of the control at 10 µM in cardiomyocytes under conditions of chronic beta-adrenergic receptor activation with little inhibitory effect upon protein kinase A activity and minimal toxicity.
Assuntos
Insuficiência Cardíaca , Proteínas Proto-Oncogênicas , Animais , Apoptose , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína 11 Semelhante a Bcl-2/farmacologia , Fibroblastos/metabolismo , Insuficiência Cardíaca/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Dihydrodipicolinate synthase (DHDPS), responsible for the first committed step of the diaminopimelate pathway for lysine biosynthesis, has become an attractive target for the development of new antibacterial and herbicidal agents. Herein, we report the discovery and exploration of the first inhibitors of E. coli DHDPS which have been identified from screening lead and are not based on substrates from the lysine biosynthesis pathway. Over 50 thiazolidinediones and related analogues have been prepared in order to thoroughly evaluate the structure-activity relationships against this enzyme of significant interest.
Assuntos
Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Hidroliases/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Hidroliases/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Tiazolidinedionas/síntese química , Tiazolidinedionas/químicaRESUMO
Bacterial thiol-disulfide oxidoreductase DsbA is essential for bacterial virulence factor assembly and has been identified as a viable antivirulence target. Herein, we report a structure-based elaboration of a benzofuran hit that bound to the active site groove of Escherichia coli DsbA. Substituted phenyl groups were installed at the 5- and 6-position of the benzofuran using Suzuki-Miyaura coupling. HSQC NMR titration experiments showed dissociation constants of this series in the high µM to low mM range and X-ray crystallography produced three co-structures, showing binding in the hydrophobic groove, comparable with that of the previously reported benzofurans. The 6-(m-methoxy)phenyl analogue (2b), which showed a promising binding pose, was chosen for elaboration from the C-2 position. The 2,6-disubstituted analogues bound to the hydrophobic region of the binding groove and the C-2 groups extended into the more polar, previously un-probed, region of the binding groove. Biochemical analysis of the 2,6-disubsituted analogues showed they inhibited DsbA oxidation activity in vitro. The results indicate the potential to develop the elaborated benzofuran series into a novel class of antivirulence compounds.
Assuntos
Benzofuranos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Benzofuranos/síntese química , Benzofuranos/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Estrutura Molecular , Isomerases de Dissulfetos de Proteínas/metabolismo , Relação Estrutura-AtividadeRESUMO
Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides with novel modes of action are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. The first class of plant DHDPS inhibitors with micromolar potency against Arabidopsis thaliana DHDPS was identified using a high-throughput chemical screen. We determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. The inhibitors also attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide-resistant weeds.
Assuntos
Arabidopsis/efeitos dos fármacos , Herbicidas/química , Herbicidas/farmacologia , Lisina/biossíntese , Hidroliases/metabolismo , Plantas Geneticamente ModificadasRESUMO
A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to EcDsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (6), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of EcDsbA. Binding affinity was calculated based on NMR studies and compounds 25 and 28 were identified as the highest affinity binders with dissociation constants (KD) of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of EcDsbA inhibitors.
Assuntos
Benzofuranos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/enzimologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Benzofuranos/síntese química , Benzofuranos/química , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.
Assuntos
Eritrócitos/efeitos dos fármacos , Isoquinolinas/síntese química , Plasmodium falciparum/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Resistência a Medicamentos/efeitos dos fármacos , Eritrócitos/parasitologia , Isoquinolinas/química , Isoquinolinas/farmacologia , Modelos Moleculares , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Mutação Puntual , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sódio , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Sequenciamento Completo do GenomaRESUMO
The electronic spectra of 2-bromoethylbenzene and its chloro and fluoro analogues have been recorded by resonant two-photon ionisation (R2PI) spectroscopy. Anti and gauche conformers have been assigned by rotational band contour analysis and IR-UV ion depletion spectroscopy in the CH region. Hydrate clusters of the anti conformers have also been observed, allowing the role of halocarbons as hydrogen bond acceptors to be examined in this context. The donor OH stretch of water bound to chlorine is red-shifted by 36 cm-1, or 39 cm-1 in the case of bromine. Although classed as weak H-bond acceptors, halocarbons are favourable acceptor sites compared to π systems. Fluorine stands out as the weakest H-bond acceptor amongst the halogens. Chlorine and bromine are also weak H-bond acceptors, but allow for more geometric lability, facilitating complimentary secondary interactions within the host molecule. Ab initio and DFT quantum chemical calculations, both harmonic and anharmonic, aid the structural assignments and analysis.
RESUMO
Dihydrodipicolinate synthase (DHDPS) is critical to the production of lysine through the diaminopimelate (DAP) pathway. Elucidation of the function, regulation and structure of this key class I aldolase has been the focus of considerable study in recent years, given that the dapA gene encoding DHDPS has been found to be essential to bacteria and plants. Allosteric inhibition by lysine is observed for DHDPS from plants and some bacterial species, the latter requiring a histidine or glutamate at position 56 (Escherichia coli numbering) over a basic amino acid. Structurally, two DHDPS monomers form the active site, which binds pyruvate and (S)-aspartate ß-semialdehyde, with most dimers further dimerising to form a tetrameric arrangement around a solvent-filled centre cavity. The architecture and behaviour of these dimer-of-dimers is explored in detail, including biophysical studies utilising analytical ultracentrifugation, small-angle X-ray scattering and macromolecular crystallography that show bacterial DHDPS tetramers adopt a head-to-head quaternary structure, compared to the back-to-back arrangement observed for plant DHDPS enzymes. Finally, the potential role of pyruvate in providing substrate-mediated stabilisation of DHDPS is considered.
RESUMO
With multidrug resistant bacteria on the rise, novel antibiotics are becoming highly sought after. In 2008, eleven compounds were identified by high throughput screening as inhibitors of BasE, a key enzyme of the non-ribosomal peptide synthetase pathway found in Acinetobacter baumannii. Herein, we describe the preparation of four structurally similar heterocyclic lead compounds from that study, including one 1,2,5-oxadiazole. A further library of 30 analogues containing the oxadiazole moiety was then generated. All compounds were screened against Acinetobacter baumannii and their minimum inhibitory concentration data is reported, with (E)-3-(2-hydroxyphenyl)-N-(4-methyl-1,2,5-oxadiazol-3-yl)acrylamide 32 found to have an MIC of 0.5mM. This work provides the foundation for further investigation of 1,2,5-oxadizoles as novel inhibitors of A. baumannii.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Oxidiazóis/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Relação Estrutura-AtividadeRESUMO
Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect. We subsequently employed our crystallographic data with bioinformatics, mutagenesis, enzyme kinetics, and microscale thermophoresis to reveal that lysine-mediated inhibition is not defined by Gram staining, but by the presence of a His or Glu at position 56 (Escherichia coli numbering). This study has unveiled the molecular determinants defining lysine-mediated allosteric inhibition of bacterial DHDPS.
Assuntos
Escherichia coli/enzimologia , Retroalimentação Fisiológica , Hidroliases/química , Legionella pneumophila/enzimologia , Lisina/química , Streptococcus pneumoniae/enzimologia , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Expressão Gênica , Hidroliases/genética , Hidroliases/metabolismo , Cinética , Legionella pneumophila/genética , Lisina/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/genética , Especificidade por SubstratoRESUMO
Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum parasites. Following each cycle of intracellular development and replication, parasites activate a cellular program to egress from their current host cell and invade a new one. The orchestration of this process critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number of central kinases. Parasite kinases are emerging as novel antimalarial targets as they have diverged sufficiently from their mammalian counterparts to allow selectable therapeutic action. Parasite protein kinase A (PfPKA) is highly expressed late in the cell cycle of the parasite blood stage and has been shown to phosphorylate a critical invasion protein, Apical Membrane Antigen 1. This enzyme could therefore be a valuable drug target so we have repurposed a substituted 4-cyano-3-methylisoquinoline that has been shown to inhibit rat PKA with the goal of targeting PfPKA. We synthesised a novel series of compounds and, although many potently inhibit the growth of chloroquine sensitive and resistant strains of P. falciparum, they were found to have minimal activity against PfPKA, indicating that they likely have another target important to parasite cytokinesis and invasion.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Desenho de Fármacos , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sequência de Aminoácidos , Antimaláricos/química , Técnicas de Química Sintética , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Avaliação Pré-Clínica de Medicamentos , Isoquinolinas/química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
A series of isoquinolines have been evaluated in a homology model of Plasmodium falciparum Protein Kinase A (PfPKA) using molecular dynamics. Synthesis of these compounds was then undertaken to investigate their structure-activity relationships. One compound was found to inhibit parasite growth in an in vitro assay and provides a lead to further develop 3-methylisoquinoline-4-carbonitriles as antimalarial compounds. Development of a potent and selective PfPKA inhibitor would provide a useful tool to shed further insight into the mechanisms enabling malaria parasites to establish infection.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Isoquinolinas/farmacologia , Nitrilas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Isoquinolinas/síntese química , Isoquinolinas/química , Estrutura Molecular , Nitrilas/síntese química , Nitrilas/química , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/farmacologia , Superóxido Dismutase/metabolismo , Vitaminas/farmacologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Células Cultivadas , Camundongos , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/química , Superóxido Dismutase/deficiência , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Termodinâmica , Vitaminas/químicaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal and incurable neurodegenerative disorder. Motor neurone degeneration can be caused by genetic mutation but the exact etiology of the disease, particularly for sporadic illness, still remains unclear. Therapeutics which target known pathogenic mechanisms involved in ALS, such as protein aggregation, oxidative stress, apoptosis, inflammation, endoplasmic reticulum stress and mitochondria dysfunction, are currently being pursued in order to provide neuroprotection which may be able to slow down, or perhaps even halt, disease progression. This present review focuses on the compounds which have been recently evaluated using the SOD1 mouse model, the most widely used preclinical model for ALS research.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/genética , Animais , Produtos Biológicos/uso terapêutico , Modelos Animais de Doenças , Descoberta de Drogas , Humanos , CamundongosRESUMO
Here, we review recent studies aimed at defining the importance of quaternary structure to a model oligomeric enzyme, dihydrodipicolinate synthase. This will illustrate the complementary and synergistic outcomes of coupling the techniques of analytical ultracentrifugation with enzyme kinetics, in vitro mutagenesis, macromolecular crystallography, small angle X-ray scattering, and molecular dynamics simulations, to demonstrate the role of subunit self-association in facilitating protein dynamics and enzyme function. This multitechnique approach has yielded new insights into the molecular evolution of protein quaternary structure.
Assuntos
Proteínas de Bactérias/química , Hidroliases/química , Proteínas de Plantas/química , Proteínas de Bactérias/isolamento & purificação , Evolução Molecular , Hidroliases/isolamento & purificação , Cinética , Simulação de Dinâmica Molecular , Proteínas de Plantas/isolamento & purificação , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Espalhamento a Baixo Ângulo , Ultracentrifugação , Difração de Raios XRESUMO
PDK1, a biological target that has attracted a large amount of attention recently, is responsible for the positive regulation of the PI3K/Akt pathway that is often activated in a large number of human cancers. A series of second-generation 2-anilino-4-substituted-7H-pyrrolopyrimidines were synthesised by installation of various functions at the 4-position of the 7H-pyrrolopyrimidine scaffold. All compounds were screened against the isolated PDK1 enzyme and dose response analysis was obtained on the best compounds of the series.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirróis/síntese química , Pirróis/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Relação Estrutura-AtividadeRESUMO
PDK1 is an important regulator of the PI3K/Akt pathway, which has been found frequently activated in a large number of human cancers. Herein we described the preparation of novel substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors. The synthesis is based around a Buchwald-Hartwig cross-coupling of various 3-bromo-6-substituted-quinolin-2(1H)-ones with three different functionalised anilines. The modular nature of the designed synthesis allowed access to a series of novel inhibitors through derivatisation of a late-stage intermediate. All compounds were screened against isolated PDK1 enzyme, with modest inhibition observed.