RESUMO
Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and ß1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (ß1/ß1B) mimetic peptide, ßadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore ßadp1's mechanism and develop novel SCN1B mimetic peptides affecting ß1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in ß1-expressing cells, we observed ßadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of ßadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and ß1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. ßadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, ßadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (ß1/ß1B) mimetic peptides are reported with the potential to modulate intercellular VGSC ß1-mediated adhesion, potentially through ß1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.
Assuntos
Miócitos Cardíacos , Peptídeos , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Peptídeos/farmacologia , Peptídeos/química , Humanos , Potenciais de Ação/efeitos dos fármacos , Adesão Celular/efeitos dos fármacosRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) strains that are resistant to all forms of penicillin have become an increasingly common and urgent problem threatening human health. They are responsible for a wide variety of infectious diseases ranging from minor skin abscesses to life-threatening severe infections. The vra operon that is conserved among S. aureus strains encodes a three-component signal transduction system (vraTSR) that is responsible for sensing and responding to cell wall stress. We developed a novel and multifaceted assay to identify compounds that potentiate the activity of oxacillin, essentially restoring efficacy of oxacillin against MRSA, and performed high-throughput screening (HTS) to identify oxacillin potentiators. HTS of 13,840 small-molecule compounds from an antimicrobial-focused Life Chemicals library, using the MRSA cell-based assay, identified three different inhibitor scaffolds. Checkerboard assays for synergy with oxacillin, reverse transcriptase PCR (RT-PCR) assays against vraR expression, and direct confirmation of interaction with VraS by surface plasmon resonance (SPR) further verified them to be viable hit compounds. A subsequent structure-activity relationship (SAR) study of the best scaffold with diverse analogs was utilized to improve potency and provides a strong foundation for further development.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Relação Estrutura-AtividadeRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.
Assuntos
Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Tioguanina/farmacologia , Cristalografia por Raios X , Difosfotransferases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tioguanina/análogos & derivados , Tioguanina/químicaRESUMO
Cardiovascular disease remains the single largest cause of natural death in the United States, with a significant cause of mortality associated with cardiac arrhythmias. Presently, options for treating and preventing myocardial electrical dysfunction, including sudden cardiac death, are limited. Recent studies have indicated that conduction of electrical activation in the heart may have an ephaptic component, wherein intercellular coupling occurs via electrochemical signaling across narrow extracellular clefts between cardiomyocytes. The perinexus is a 100-200â¯nm-wide stretch of closely apposed membrane directly adjacent to connexin 43 gap junctions. Electron and super-resolution microscopy studies, as well as biochemical analyses, have provided evidence that perinexal nanodomains may be candidate structures for facilitating ephaptic coupling. This work has included characterization of the perinexus as a region of close inter-membrane contact between cardiomyocytes (<30â¯nm) containing dense clusters of voltage-gated sodium channels. Here, we review what is known about perinexal structure and function and the potential that the perinexus may have novel and pivotal roles in disorders of cardiac conduction. Of particular interest is the prospect that cell adhesion mediated by the cardiac sodium channel ß subunit (Scn1b) may be a novel anti-arrhythmic target.
Assuntos
Antiarrítmicos/farmacologia , Junções Comunicantes/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiologia , Terapia de Alvo Molecular/métodos , Potenciais de Ação/efeitos dos fármacos , Animais , Junções Comunicantes/metabolismo , Humanos , Miocárdio/citologia , Miocárdio/metabolismoRESUMO
Background α Carboxyl terminus 1 (αCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study αCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an α-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.
Assuntos
Conexina 43/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/metabolismo , Fragmentos de Peptídeos/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/efeitos dos fármacos , Animais , Simulação por Computador , Conexina 43/metabolismo , Camundongos , Microscopia Confocal , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/patologia , Fosforilação , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that ß1(SCN1B) -mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential ß1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, ßadp1, potently and selectively inhibited ß1-mediated adhesion, in electric cell-substrate impedance sensing studies. ßadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, ßadp1 precipitated arrhythmogenic conduction slowing. In summary, ß1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.
Assuntos
Arritmias Cardíacas/tratamento farmacológico , Comunicação Celular/genética , Junções Comunicantes/ultraestrutura , Miócitos Cardíacos/fisiologia , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Adesão Celular/genética , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Biologia Computacional , Impedância Elétrica , Junções Comunicantes/fisiologia , Cobaias , Humanos , Camundongos , Modelos Cardiovasculares , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Técnicas de Patch-Clamp , Peptídeos/química , Sódio/metabolismo , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genéticaRESUMO
Pediatric tuberculosis is an underappreciated global epidemic estimated to afflict around half a million children worldwide. This problem has historically been overlooked, due in part to their low social status and the difficulty in diagnosis of tuberculosis in children. Children are more susceptible to tuberculosis infection and disease progression, including rapid dissemination into extrapulmonary infection sites. Treatment of pediatric tuberculosis infections has been traditionally built around agents used to treat the adult disease, but the disease pathology, drug pharmacokinetics and the safety window in children differs from the adult disease. This produces additional concerns for drug discovery and development of new agents. This review examines: (i) the safety concerns for current front and second line agents used to treat complex drug resistant infections and how this knowledge can be used to identify, prioritize and dose agents that may be better tolerated in pediatric populations; and (ii) the chemistry and suitability of new drugs in the clinical development pipeline for tuberculosis for the treatment of pediatric infections indicating several new agents may offer significant improvements for the treatment of multi-drug resistant tuberculosis in children.
Assuntos
Antituberculosos/uso terapêutico , Descoberta de Drogas , Mycobacterium/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Animais , Antituberculosos/efeitos adversos , Antituberculosos/química , Antituberculosos/farmacologia , Criança , Descoberta de Drogas/métodos , Farmacorresistência Bacteriana , HumanosRESUMO
Inadequate dosing and incomplete treatment regimens, coupled with the ability of the tuberculosis bacilli to cause latent infections that are tolerant of currently used drugs, have fueled the rise of multidrug-resistant tuberculosis (MDR-TB). Treatment of MDR-TB infections is a major clinical challenge that has few viable or effective solutions; therefore patients face a poor prognosis and years of treatment. This review focuses on emerging drug classes that have the potential for treating MDR-TB and highlights their particular strengths as leads including their mode of action, in vivo efficacy, and key medicinal chemistry properties. Examples include the newly approved drugs bedaquiline and delaminid, and other agents in clinical and late preclinical development pipeline for the treatment of MDR-TB. Herein, we discuss the challenges to developing drugs to treat tuberculosis and how the field has adapted to these difficulties, with an emphasis on drug discovery approaches that might produce more effective agents and treatment regimens.