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Adv Exp Med Biol ; 1131: 27-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646506


Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.

Bioensaio , Canais Iônicos , Receptores Acoplados a Proteínas-G , Animais , Bioensaio/tendências , Descoberta de Drogas , Corantes Fluorescentes/metabolismo , Humanos , Canais Iônicos/análise , Receptores Acoplados a Proteínas-G/análise
PLoS One ; 14(3): e0213751, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30856233


Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 µM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 µM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Neurosci Lett ; 679: 4-14, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-29425731


Toxins have been used as tools for decades to study the structure and function of neuronal ion channels and receptors. The biological origin of these toxins varies from single cell organisms, including bacteria and algae, to complex multicellular organisms, including a wide variety of plants and venomous animals. Toxins are a structurally and functionally diverse group of compounds that often modulate neuronal function by interacting with an ion channel or receptor. Many of these toxins display high affinity and exquisite selectivity, making them valuable tools to probe the structure and function of neuronal ion channels and receptors. This review article provides an overview of the experimental techniques used to assess the effects that toxins have on neuronal function, as well as discussion on toxins that have been used as tools, with a focus on toxins that target voltage-gated and ligand-gated ion channels.

Adv Pharmacol ; 79: 67-116, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28528674


Venomous animals including cone snails, spiders, scorpions, anemones, and snakes have evolved a myriad of components in their venoms that target the opening and/or closing of voltage-gated sodium channels to cause devastating effects on the neuromuscular systems of predators and prey. These venom peptides, through design and serendipity, have not only contributed significantly to our understanding of sodium channel pharmacology and structure, but they also represent some of the most phyla- and isoform-selective molecules that are useful as valuable tool compounds and drug leads. Here, we review our understanding of the basic function of mammalian voltage-gated sodium channel isoforms as well as the pharmacology of venom peptides that act at these key transmembrane proteins.

Peptídeos/farmacologia , Canais de Sódio/metabolismo , Peçonhas/farmacologia , Sequência de Aminoácidos , Animais , Humanos , Canais de Sódio Disparados por Voltagem/metabolismo
Contemp Clin Trials ; 41: 313-24, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25583272


UNLABELLED: Population aging is associated with a higher prevalence of chronic health conditions. Previous studies have shown that older persons, specifically those with chronic conditions, often lack sufficient knowledge about their condition and thus frequently have poor self-care skills. Efforts to increase general health screenings and improve access to chronic condition management resources are hampered by a lack of disease and health awareness. Self-Care for Older People (SCOPE) study, a cluster randomized controlled trial in Singapore, was designed to evaluate the impact of a self-care program for chronic disease awareness and management of specific health measures and quality of life of older people over eighteen months. SCOPE provided self-care education targeted at older people with low income and low education in order to improve health-related knowledge. A total of 378 low-income older people with no or minimal disability, defined as having difficulty in one or more activities of daily living (ADL), were recruited from senior activity centers. The measurements taken included self-reported health conditions, health and disease knowledge questions, and biomarkers (HbA1c, blood pressure, peak expiratory flow, lipid panel, albumin, and creatinine). SCOPE was also designed to provide information for policy makers on chronic disease burden and healthcare facility utilization among community-dwelling older adults. TRIAL REGISTRATION NUMBER: NCT01672177.

Atividades Cotidianas , Diabetes Mellitus/reabilitação , Hipertensão/reabilitação , Educação de Pacientes como Assunto/métodos , Doença Pulmonar Obstrutiva Crônica/reabilitação , Autocuidado/métodos , Idoso , Doença Crônica , Humanos , Pessoa de Meia-Idade , Pobreza , Qualidade de Vida , Singapura