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BACKGROUND AND PURPOSE: Inhibitors of voltage-gated sodium channels (NaVs) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Nav channels as a means of informing the development of improved antiseizure drugs. EXPERIMENTAL APPROACH: We created a series of compounds with diverse selectivity profiles enabling block of NaV1.6 alone or together with NaV1.2. These novel NaV inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding NaV1.6) and in wild-type mice. KEY RESULTS: Pharmacologic inhibition of NaV1.6 in Scn8aN1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. NaV1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS NaV isoforms. CONCLUSIONS AND IMPLICATIONS: Our data suggest NaV1.6 inhibition is a driver of efficacy for NaV inhibitor anti-seizure medicines. Sparing the NaV1.1 channels of inhibitory interneurons did not compromise efficacy. Selective NaV1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies.
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Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.
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Epilepsia , Canais de Sódio Disparados por Voltagem , Humanos , Neurônios/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Epilepsia/metabolismo , Encéfalo/metabolismo , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Potenciais de Ação/fisiologiaRESUMO
BACKGROUND: The natural history of primary sclerosing cholangitis (PSC) among African Americans (AA) is not well understood. METHODS: Transplant-free survival and hepatic decompensation-free survival were assessed using a retrospective research registry from 16 centers throughout North America. Patients with PSC alive without liver transplantation after 2008 were included. Diagnostic delay was defined from the first abnormal liver test to the first abnormal cholangiogram/liver biopsy. Socioeconomic status was imputed by the Zip code. RESULTS: Among 850 patients, 661 (77.8%) were non-Hispanic Whites (NHWs), and 85 (10.0%) were AA. There were no significant differences by race in age at diagnosis, sex, or PSC type. Inflammatory bowel disease was more common in NHWs (75.8% vs. 51.8% p=0.0001). The baseline (median, IQR) Amsterdam-Oxford Model score was lower in NHWs (14.3, 13.4-15.2 vs. 15.1, 14.1-15.7, p=0.002), but Mayo risk score (0.03, -0.8 to 1.1 vs. 0.02, -0.7 to 1.0, p=0.83), Model for End-stage Liver Disease (5.9, 2.8-10.7 vs. 6.4, 2.6-10.4, p=0.95), and cirrhosis (27.4% vs. 27.1%, p=0.95) did not differ. Race was not associated with hepatic decompensation, and after adjusting for clinical variables, neither race nor socioeconomic status was associated with transplant-free survival. Variables independently associated with death/liver transplant (HR, 95% CI) included age at diagnosis (1.04, 1.02-1.06, p<0.0001), total bilirubin (1.06, 1.04-1.08, p<0.0001), and albumin (0.44, 0.33-0.61, p<0.0001). AA race did not affect the performance of prognostic models. CONCLUSIONS: AA patients with PSC have a lower rate of inflammatory bowel disease but similar progression to hepatic decompensation and liver transplant/death compared to NHWs.
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Colangite Esclerosante , Doença Hepática Terminal , Doenças Inflamatórias Intestinais , Humanos , Estudos Retrospectivos , Colangite Esclerosante/diagnóstico , Negro ou Afro-Americano , Diagnóstico Tardio , Índice de Gravidade de Doença , Doenças Inflamatórias Intestinais/complicaçõesRESUMO
BACKGROUND: SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia. METHODS: In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identified via exome sequencing. RESULTS: Clinical findings ranged from normal development with well-controlled epilepsy to significant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4. CONCLUSIONS: This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant.
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Disfunção Cognitiva , Epilepsia Generalizada , Epilepsia , Humanos , Epilepsia/genética , Genótipo , Fenótipo , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genéticaRESUMO
Background: SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia. Methods: In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identified via exome sequencing. Results: Clinical findings ranged from normal development with well-controlled epilepsy to significant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4. Conclusions: This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant.
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Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.
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Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Triptofano , Peixe-Zebra , MutaçãoRESUMO
The potential for the use of artificial intelligence in developing lethal autonomous weapons systems (LAWS) has received a good deal of attention from ethicists. Lines of argument in favor of and against developing and deploying LAWS have already become hardened. In this paper, I examine one strategy for skirting these familiar positions, namely to base an anti-LAWS argument not on claims that LAWS inevitably fail to respect human dignity, but on a different kind of respect, namely respect for public opinion and conventional attitudes (which Robert Sparrow claims are strongly anti-LAWS). My conclusion is that this sort of respect for conventional attitudes does provide some reason for actions and policies, but that it is actually a fairly weak form of respect, that is often override by more direct concerns about respect for humanity or dignity. By doing this, I explain the intuitive force of the claim that one should not disregard public attitudes, but also justify assigning a relatively weak role when other kinds of respect are involved.
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Infants are frequently supplemented with iron to prevent iron deficiency, but iron supplements may have adverse effects on infant health. Although iron supplements can be highly effective at improving iron status and preventing iron deficiency anemia, iron may adversely affect growth and development, and may increase risk for certain infections. Several reviews exist in this area; however, none has fully summarized all reported outcomes of iron supplementation during infancy. In this review, we summarize the risks and benefits of iron supplementation as they have been reported in controlled studies and in relevant animal models. Additionally, we discuss the mechanisms that may underly beneficial and adverse effects.
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Anemia Ferropriva , Deficiências de Ferro , Animais , Ferro/efeitos adversos , Anemia Ferropriva/prevenção & controle , Suplementos Nutricionais/efeitos adversos , Medição de RiscoRESUMO
NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.
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Epilepsia , Canal de Sódio Disparado por Voltagem NAV1.6 , Animais , Mutação com Ganho de Função , Camundongos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Ratos , Sódio , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
Background: COVID-19 elimination measures, including border closures have been applied in New Zealand. We have modelled the potential effect of vaccination programmes for opening borders. Methods: We used a deterministic age-stratified Susceptible, Exposed, Infectious, Recovered (SEIR) model. We minimised spread by varying the age-stratified vaccine allocation to find the minimum herd immunity requirements (the effective reproduction number Reff<1 with closed borders) under various vaccine effectiveness (VE) scenarios and R0 values. We ran two-year open-border simulations for two vaccine strategies: minimising Reff and targeting high-risk groups. Findings: Targeting of high-risk groups will result in lower hospitalisations and deaths in most scenarios. Reaching the herd immunity threshold (HIT) with a vaccine of 90% VE against disease and 80% VE against infection requires at least 86â¢5% total population uptake for R0=4â¢5 (with high vaccination coverage for 30-49-year-olds) and 98â¢1% uptake for R0=6. In a two-year open-border scenario with 10 overseas cases daily and 90% total population vaccine uptake (including 0-15 year olds) with the same vaccine, the strategy of targeting high-risk groups is close to achieving HIT, with an estimated 11,400 total hospitalisations (peak 324 active and 36 new daily cases in hospitals), and 1,030 total deaths. Interpretation: Targeting high-risk groups for vaccination will result in fewer hospitalisations and deaths with open borders compared to targeting reduced transmission. With a highly effective vaccine and a high total uptake, opening borders will result in increasing cases, hospitalisations, and deaths. Other public health and social measures will still be required as part of an effective pandemic response. Funding: This project was funded by the Health Research Council [20/1018]. Research in context.
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We describe the synthesis and biological evaluation of a series of novel aryl sulfonamides that exhibit potent inhibition of NaV1.5. Unlike local anesthetics that are currently used for treatment of Long QT Syndrome 3 (LQT-3), the most potent compound (-)-6 in this series shows high selectivity over hERG and other cardiac ion channels and has a low brain to plasma ratio to minimize CNS side effects. Compound (-)-6 is also effective inshortening prolonged action potential durations (APDs) in a pharmacological model of LQT-3 syndrome in pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Unlike most aryl sulfonamide NaV inhibitors that bind to the channel voltage sensors, these NaV1.5 inhibitors bind to the local anesthetic binding site in the central pore of the channel.
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Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Sulfonamidas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.
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Azetidinas/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Azetidinas/química , Azetidinas/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Células Cultivadas , Células HEK293 , Humanos , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacologia , Ratos Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1-1.7 currents, with an IC50 of 1.9-3.8 µm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of â¼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.
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Canabidiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.1/química , Canal de Sódio Disparado por Voltagem NAV1.6/química , Neurônios/patologia , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/química , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Sódio/metabolismo , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genética , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismoAssuntos
Mycobacterium tuberculosis , Tuberculose , África , Estudos Epidemiológicos , Europa (Continente) , HumanosRESUMO
Mutations of DNA repair pathways contribute to tumorigenesis and provide a therapeutic target for synthetic lethal interactions in tumor cells. Given that tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs stalled topoisomerase-I DNA complexes, we hypothesized that inhibition of Tdp1 has synthetic lethal effects in some cancers. To test this, we screened tumor arrays for Tdp1 expression and observed that Tdp1 is expressed in many tumors, including more than 90% of human breast tumors. Subsequent chemical screening identified putative Tdp1 inhibitors. Treatment of control human mammary epithelial cells and the breast cancer cell line MCF-7 with compound CD00509 preferentially sensitized MCF-7 cells to camptothecin and decreased cell proliferation 25% more than camptothecin treatment alone. This suggests that CD00509 specifically targeted Tdp1 in vitro, and CD00509 increased the sensitivity of wild-type murine embryonic fibroblasts (MEFs) to camptothecin to a degree comparable to that of Tdp1(-/-) MEFs. In addition, consistent with poly ADP-ribose polymerase-1 (PARP-1) collaborating with Tdp1 in DNA repair, combined Tdp1 and PARP-1 inhibition was more detrimental to MCF-7 cells than either treatment alone, whereas the combination was not additively harmful to control mammary cells. We conclude that targeting Tdp1 in anticancer therapy preferentially enhances the sensitivity of some breast cancer cells to camptothecin and may be an effective adjuvant for breast cancer therapy.
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Ensaios de Triagem em Larga Escala/métodos , Neoplasias/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Piranos/farmacologia , Tiobarbitúricos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Dano ao DNA/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Técnicas In Vitro , Células MCF-7/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Análise Serial de Tecidos , Inibidores da Topoisomerase I/farmacologiaRESUMO
The stigmatization of some groups of people, whether for some characteristic they possess or some behavior they engage in, will initially strike most of us as wrong. For many years, academic work in public health, which focused mainly on the stigmatization of HIV-positive individuals, reinforced this natural reaction to stigmatization, by pointing out the negative health effects of stigmatization. But more recently, the apparent success of anti-smoking campaigns which employ stigmatization of smokers has raised questions about whether stigmatization may sometimes be justified, because of its positive effects on public health. Discussion of the issue so far has focused on consequences, and on some Kantian considerations regarding the status of the stigmatized. In this article, I argue that further Kantian considerations regarding the treatment of the general public (the potential stigmatizers) also count against any public health policy involving stigmatization. Attempts to encourage stigmatization are likely to fail to appeal to the rational decision-making abilities of the general public, and the creation of stigmatized groups (even if they are stigmatized for their voluntary behavior) is an obstacle to the self-improvement of members of the general public.
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Comportamento de Escolha , Política de Saúde/tendências , Princípios Morais , Preconceito , Saúde Pública/ética , Opinião Pública , Fumar , Estereotipagem , Análise Ética , Teoria Ética , Infecções por HIV/psicologia , Humanos , Obrigações Morais , Preconceito/ética , Preconceito/tendências , Saúde Pública/métodos , Saúde Pública/tendências , Política Pública/tendências , Fumar/psicologiaRESUMO
Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.
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Benzoatos/farmacologia , HDL-Colesterol/sangue , HDL-Colesterol/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Lipase/antagonistas & inibidores , Pirrolidinas/farmacologia , Animais , Benzoatos/síntese química , Benzoatos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Lipase/deficiência , Lipase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Pirrolidinas/síntese química , Pirrolidinas/química , Relação Estrutura-AtividadeRESUMO
Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.
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Marcação por Isótopo/métodos , Metaloproteinases da Matriz/metabolismo , Proteômica/métodos , Animais , Células Cultivadas , Cromatografia Líquida , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Proteínas/análise , Reprodutibilidade dos Testes , Estatística como Assunto , Especificidade por SubstratoRESUMO
The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.
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Membrana Celular/química , Membrana Celular/enzimologia , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Cromatografia Líquida , Feminino , Humanos , Marcação por Isótopo , Espectrometria de Massas , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/genética , Metabolômica/métodos , Proteínas Mutantes/metabolismo , Peptídeo Hidrolases/genética , Reprodutibilidade dos Testes , Especificidade por Substrato , TransfecçãoRESUMO
Through the activity of macrophage-specific matrix metalloproteinase-12 (MMP-12), we found that macrophages dampen the lipopolysaccharide (LPS)-induced influx of polymorphonuclear leukocytes (PMNs)-thus providing a new mechanism for the termination of PMN recruitment in acute inflammation. MMP-12 specifically cleaves human ELR(+) CXC chemokines (CXCL1, -2, -3, -5, and -8) at E-LR, the critical receptor-binding motif or, for CXCL6, carboxyl-terminal to it. Murine (m) MMP-12 also cleaves mCXCL1, -2, and -3 at E-LR. MMP-12-cleaved mCXCL2 (macrophage-inflammatory protein-2 [MIP-2]) and mCXCL3 (dendritic cell inflammatory protein-1 [DCIP-1]) lost chemotactic activity. Furthermore, MMP-12 processed and inactivated monocyte chemotactic proteins CCL2, -7, -8, and -13 at position 4-5 generating CCR antagonists. Indeed, PMNs and macrophages in bronchoalveolar lavage fluid were significantly increased 72 hours after intranasal instillation of LPS in Mmp12(-/-) mice compared with wild type. Specificity occurred at 2 levels. Macrophage MMP-1 and MMP-9 did not cleave in the ELR motif. Second, unlike human ELR(+)CXC chemokines, mCXCL5 (LPS-induced CXC chemokine [LIX]) was not inactivated. Rather, mMMP-12 cleavage at Ser4-Val5 activated the chemokine, promoting enhanced PMN early infiltration in wild-type mice compared with Mmp12(-/-) mice 8 hours after LPS challenge in air pouches. We propose that the macrophage, specifically through MMP-12, assists in orchestrating the regulation of acute inflammatory responses by precise proteolysis of ELR(+)CXC and CC chemokines.