RESUMO
The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
Assuntos
Alanina/análogos & derivados , Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca mulatta/virologia , Ribonucleotídeos/uso terapêutico , Monofosfato de Adenosina/análogos & derivados , Alanina/farmacocinética , Alanina/farmacologia , Alanina/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular Tumoral , Ebolavirus/efeitos dos fármacos , Feminino , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Ribonucleotídeos/farmacocinética , Ribonucleotídeos/farmacologiaRESUMO
BACKGROUND: Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from 'host' cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy. METHODS: After production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP1 concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP1 protein. Purified recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm. RESULTS: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP1 with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6 %. Unlike western blot values, the LC-HRMS quantitation of GP1 in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice. CONCLUSIONS: This method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1. By monitoring variability in GP1 content, the eVLP production process can be optimized, and the total amount of GP1 needed to confer protection accurately determined.
RESUMO
Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure-activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Neurotoxinas/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Toxinas Botulínicas Tipo A/química , Botulismo/tratamento farmacológico , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neurotoxinas/química , Nitrilas/química , Nitrilas/farmacologia , Relação Estrutura-AtividadeRESUMO
Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Ebolavirus/fisiologia , Interações Hospedeiro-Patógeno , Nucleoproteínas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , DNA Helicases/genética , Ebolavirus/genética , Técnicas de Silenciamento de Genes , Genoma Viral , Humanos , Nucleoproteínas/genética , Ligação Proteica , RNA Interferente PequenoRESUMO
Age-associated mitochondrial dysfunction is a major source of reactive oxygen species (ROS) and oxidative modification to proteins. Mitochondrial electron transport chain (ETC) complexes I and III are the sites of ROS production and we hypothesize that proteins of the ETC complexes are primary targets of ROS-mediated modification which impairs their structure and function. The pectoralis, primarily an aerobic red muscle, and quadriceps, primarily an anaerobic white muscle, have different rates of respiration and oxygen-carrying capacity, and hence, different rates of ROS production. This raises the question of whether these muscles exhibit different levels of oxidative protein modification. Our studies reveal that the pectoralis shows a dramatic age-related decline in almost all complex activities that correlates with increased oxidative modification. Similar complex proteins were modified in the quadriceps, at a significantly lower level with less change in enzyme and ETC coupling function. We postulate that mitochondrial ROS causes damage to specific ETC subunits which increases with age and leads to further mitochondrial dysfunction. We conclude that physiological characteristics of the pectoralis vs quadriceps may play a role in age-associated rate of mitochondrial dysfunction and in the decline in tissue function.
Assuntos
Envelhecimento/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Animais , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The endoplasmic reticulum (ER) is a target for endogenously generated reactive oxygen species (ROS) during aging. We have previously shown that the ER chaperones, protein disulfide isomerase (PDI) and immunoglobulin heavy chain binding protein (BiP), are oxidatively modified within the livers of aged mice. In this study we assess the functional consequences of the age-dependent oxidation of these two proteins. Specific activity measurements, performed on purified protein samples obtained from young and aged mouse livers, show definitive decreases in BiP ATPase activity and dramatic reductions in PDI enzymatic activity with age. Overall, these results suggest that protein folding and other activities mediated through PDI and BiP are diminished during aging. Furthermore, the relative loss of these chaperone-like activities could directly contribute to the age-dependent accumulation of misfolded proteins, a characteristic of the aging phenotype.
Assuntos
Proteínas de Choque Térmico/metabolismo , Fígado/fisiologia , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Envelhecimento/metabolismo , Animais , Regulação para Baixo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.
Assuntos
Envelhecimento/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Rim/enzimologia , Mitocôndrias/enzimologia , Estresse Oxidativo , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons/análise , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análiseRESUMO
Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Francisella tularensis/genética , Mutação/genética , Açúcares Ácidos/metabolismo , Tularemia/microbiologia , Animais , Ciprofloxacina/farmacologia , Francisella tularensis/efeitos dos fármacos , Francisella tularensis/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Profilaxia Pós-Exposição/métodos , Virulência/genéticaRESUMO
INTRODUCTION: Botulinum neurotoxins (BoNTs) are among the most toxic of known biological molecules and function as acetylcholine release inhibitors and neuromuscular blocking agents. Paradoxically, these properties also make them valuable therapeutic agents for the treatment of movement disorders, urological conditions and hypersecretory disorders. Greater understanding of their molecular mechanism of action and advances in protein engineering has led to significant efforts to improve and expand their function with a view towards broadening their therapeutic potential. AREAS COVERED: Searches of Espacenet and Google Patent have revealed a number of patents related to BoNTs. This review will focus on novel therapeutic uses and formulations disclosed during 2012 - 2014. The seven patents discussed will include nanoformulations of FDA-approved BoNTs, additional BoNT subtypes and novel BoNT variants and chimeras created through protein engineering. Supporting patents and related publications are also briefly discussed. EXPERT OPINION: The clinical and commercial success of BoNTs has prompted investigation into novel BoNTs or BoNT-mediated chimeras with promising in vitro results. Distinct strategies including the use of nanoformulations and targeted delivery have been implemented to identify new indication and improved functionality. Greater understanding of their systemic exposure, efficacy and safety profiles will be required for further development.
Assuntos
Toxinas Botulínicas/administração & dosagem , Sistemas de Liberação de Medicamentos , Neurotoxinas/administração & dosagem , Animais , Toxinas Botulínicas/efeitos adversos , Toxinas Botulínicas/uso terapêutico , Aprovação de Drogas , Humanos , Nanoestruturas , Neurotoxinas/efeitos adversos , Neurotoxinas/uso terapêutico , Patentes como Assunto , Engenharia de Proteínas/métodos , Estados Unidos , United States Food and Drug AdministrationRESUMO
There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.
Assuntos
Toxinas Botulínicas/toxicidade , Células-Tronco Embrionárias/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neurônios Motores/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Toxinas Botulínicas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Neurônios Motores/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteínas SNARE/metabolismoRESUMO
Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins' proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin's enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.
Assuntos
Toxinas Botulínicas/toxicidade , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Proteólise/efeitos dos fármacos , SorogrupoRESUMO
Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.
Assuntos
Proteínas de Ligação a DNA/química , Metano/análogos & derivados , Oligonucleotídeos/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Hidrocarbonetos , Metano/química , Oligonucleotídeos/química , Mapeamento de Peptídeos , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/análise , Subunidades Proteicas/química , Proteína de Replicação A , Análise EspectralRESUMO
Significantly more potent second generation 4-amino-7-chloroquinoline (4,7-ACQ) based inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain were synthesized. Introducing an amino group at the C(3) position of the cholate component markedly increased potency (IC50 values for such derivatives ranged from 0.81 to 2.27 µM). Two additional subclasses were prepared: bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives; both classes provided inhibitors with nanomolar-range potencies (e.g., the Ki of compound 67 is 0.10 µM). During BoNT/A challenge using primary neurons, select derivatives protected SNAP-25 by up to 89%. Docking simulations were performed to rationalize the compounds' in vitro potencies. In addition to specific residue contacts, coordination of the enzyme's catalytic zinc and expulsion of the enzyme's catalytic water were a consistent theme. With respect to antimalarial activity, the compounds provided better IC90 activities against chloroquine resistant (CQR) malaria than CQ, and seven compounds were more active than mefloquine against CQR strain W2.
Assuntos
Aminoquinolinas/síntese química , Antimaláricos/síntese química , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Metaloproteases/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteases/síntese química , Aminoquinolinas/farmacologia , Animais , Antimaláricos/farmacologia , Embrião de Galinha , Cloroquina/farmacologia , Resistência a Medicamentos , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Relação Estrutura-AtividadeRESUMO
Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Proteômica , RNA Interferente Pequeno , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , Espectrometria de Massas em Tandem , Proteínas Virais/genética , Vírion/genéticaRESUMO
Structurally simplified analogues of dual antimalarial and botulinum neurotoxin serotype A light chain (BoNT/A LC) inhibitor bis-aminoquinoline (1) were prepared. New compounds were designed to improve ligand efficiency while maintaining or exceeding the inhibitory potency of 1. Three of the new compounds are more active than 1 against both indications. Metabolically, the new inhibitors are relatively stable and nontoxic. 12, 14, and 15 are more potent BoNT/A LC inhibitors than 1. Additionally, 15 has excellent in vitro antimalarial efficacy, with IC90 values ranging from 4.45 to 12.11 nM against five Plasmodium falciparum (P.f.) strains: W2, D6, C235, C2A, and C2B. The results indicate that the same level of inhibitory efficacy provided by 1 can be retained/exceeded with less structural complexity. 12, 14, and 15 provide new platforms for the development of more potent dual BoNT/A LC and P.f. inhibitors adhering to generally accepted chemical properties associated with the druggability of synthetic molecules.
Assuntos
Antimaláricos/síntese química , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Quinolinas/síntese química , Antimaláricos/farmacologia , Células Hep G2 , Humanos , Ligantes , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The age-associated decline in tissue function has been attributed to ROS-mediated oxidative damage due to mitochondrial dysfunction. The long-lived Ames dwarf mouse exhibits resistance to oxidative stress, a physiological characteristic of longevity. It is not known, however, whether there are differences in the electron transport chain (ETC) functions in Ames tissues that are associated with their longevity. In these studies we analyzed enzyme activities of ETC complexes, CI-CV and the coupled CI-CII and CII-CIII activities of mitochondria from several tissues of young, middle aged and old Ames dwarf mice and their corresponding wild type controls to identify potential mitochondrial prolongevity functions. Our studies indicate that post-mitotic heart and skeletal muscle from Ames and wild-type mice show similar changes in ETC complex activities with aging, with the exception of complex IV. Furthermore, the kidney, a slowly proliferating tissue, shows dramatic differences in ETC functions unique to the Ames mice. Our data show that there are tissue specific mitochondrial functions that are characteristic of certain tissues of the long-lived Ames mouse. We propose that this may be a factor in the determination of extended lifespan of dwarf mice.
Assuntos
Envelhecimento/fisiologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica/fisiologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Animais , Nanismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Rim/enzimologia , Camundongos , Camundongos Endogâmicos , MutaçãoRESUMO
Botulinum neurotoxins (BoNTs) comprise seven distinct serotypes that inhibit the release of neurotransmitter across neuromuscular junctions, resulting in potentially fatal flaccid paralysis. BoNT serotype A (BoNT/A), which targets synaptosomal-associated protein of 25kDa (SNAP-25), is particularly long-lived within neurons and requires a longer time for recovery of neuromuscular function. There are currently no treatments available to counteract BoNT/A after it has entered the neuronal cytosol. In this study, we examined the ability of small molecule non-peptidic inhibitors (SMNPIs) to prevent SNAP-25 cleavage post-intoxication of neurons. The progressive cleavage of SNAP-25 observed over 5 h following 1 h BoNT/A intoxication was prevented by addition of SMNPIs. In contrast, anti-BoNT/A neutralizing antibodies that strongly inhibited SNAP-25 cleavage when added during intoxication were completely ineffective when added post-intoxication. Although Bafilomycin A1, which blocks entry of BoNT/A into the cytosol by preventing endosomal acidification, inhibited SNAP-25 cleavage post-intoxication, the degree of inhibition was significantly reduced versus addition both during and after intoxication. Post-intoxication application of SMNPIs, on the other hand, was nearly as effective as application both during and after intoxication. Taken together, the results indicate that competitive SMNPIs of BoNT/A light chain can be effective within neurons post-intoxication.
Assuntos
Aconitina/análogos & derivados , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Imidazóis/farmacologia , Neurônios Motores/efeitos dos fármacos , Ftalimidas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Aconitina/administração & dosagem , Aconitina/química , Aconitina/farmacologia , Animais , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Galinha , Citosol/efeitos dos fármacos , Citosol/metabolismo , Imidazóis/administração & dosagem , Imidazóis/química , Macrolídeos/administração & dosagem , Macrolídeos/farmacologia , Estrutura Molecular , Neurônios Motores/metabolismo , Ftalimidas/administração & dosagem , Ftalimidas/química , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Botulinum Neurotoxins (BoNTs) are used therapeutically and in cosmetics, providing potential for bioterrorist activity, thus driving the search for small-molecule BoNT inhibitors. This report describes a 70,000-compound screen for inhibition of BoNT/A using a FRET assay to detect proteolysis of a peptide substrate. Hits were confirmed, followed by evaluation to determine compound specificity. Inhibitors fell into three main chemical classes, and on the basis of potency and specificity of inhibition, the activities of two chemotypes were examined further. Compounds exhibited specificity for BoNT/A LC inhibition with respect to other metalloproteases and displayed activity in a neuronal assay for botulinum intoxication.
RESUMO
Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however, due to the difficulty and poor efficiency of the procedures required to isolate the cells, they are not suitable for high-throughput drug screening assays. Here, we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable, reproducible, and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally, we demonstrated that several BoNT/A inhibitors protected SNAP-25, the BoNT/A substrate, in the ES-derived motoneuron system. Furthermore, this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics.
Assuntos
Antitoxina Botulínica/farmacologia , Toxinas Botulínicas/antagonistas & inibidores , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Neurônios Motores/efeitos dos fármacos , Animais , Toxinas Botulínicas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/instrumentação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
A 1,7-bis(alkylamino)diazachrysene-based small molecule was previously identified as an inhibitor of the botulinum neurotoxin serotype A light chain metalloprotease. Subsequently, a variety of derivatives of this chemotype were synthesized to develop structure-activity relationships, and all are inhibitors of the BoNT/A LC. Three-dimensional analyses indicated that half of the originally discovered 1,7-DAAC structure superimposed well with 4-amino-7-chloroquinoline-based antimalarial agents. This observation led to the discovery that several of the 1,7-DAAC derivatives are potent in vitro inhibitors of Plasmodium falciparum and, in general, are more efficacious against CQ-resistant strains than against CQ-susceptible strains. In addition, by inhibiting ß-hematin formation, the most efficacious 1,7-DAAC-based antimalarials employ a mechanism of action analogous to that of 4,7-ACQ-based antimalarials and are well tolerated by normal cells. One candidate was also effective when administered orally in a rodent-based malaria model. Finally, the 1,7-DAAC-based derivatives were examined for Ebola filovirus inhibition in an assay employing Vero76 cells, and three provided promising antiviral activities and acceptably low toxicities.