RESUMO
BACKGROUND/OBJECTIVES: Australian data comparing biologic treatments for moderate to severe chronic plaque psoriasis are lacking. We compared persistence on therapy across four biologic therapies (adalimumab, guselkumab, secukinumab and ustekinumab) used to treat chronic plaque psoriasis. The impact of prior biologic use on persistence was also investigated. METHODS: This retrospective cohort analysis of the Pharmaceutical Benefits Scheme (PBS) 10% sample included data from adult patients prescribed ≥1 biologic of interest by a dermatologist from 1 September 2015 to 31 December 2021. Persistence was defined as continued use until 180 days without a prescription. The index date was the date of the first claim of the biologic. Persistence was evaluated using Kaplan-Meier methods, log-rank tests, adjusted analyses using Cox's regressions, and propensity score matching. RESULTS: In total, 878 patients, with 1131 index prescriptions, were included. Guselkumab median persistence was not reached in the study period (PBS listed from February 2019). In the adjusted analysis, persistence to guselkumab was significantly greater than to adalimumab (n = 105; median 16 months, HR 2.71 (95% CI 1.94-3.8), p < 0.001), ustekinumab (n = 336; median 19 months, HR 2.91 (95% CI 2.22-3.82), p < 0.001) and secukinumab (n = 305; median 30 months, HR 1.8 (95% CI 1.36-2.38), p < 0.001). Bio-naïve patients had longer persistence on treatment than bio-experienced patients. CONCLUSIONS: The nationally representative PBS dataset can provide real-world insights into the persistence on biologic therapies for psoriasis in Australia, where eligibility criteria for reimbursed treatment are stringent. Persistence is an indirect marker of sustained treatment effectiveness and tolerability. Both unadjusted and adjusted analyses found longer persistence for guselkumab compared to adalimumab, secukinumab or ustekinumab.
Assuntos
Produtos Biológicos , Psoríase , Adulto , Humanos , Adalimumab/uso terapêutico , Ustekinumab/uso terapêutico , Estudos Retrospectivos , Austrália , Psoríase/tratamento farmacológico , Resultado do Tratamento , Terapia Biológica , Produtos Biológicos/uso terapêuticoRESUMO
OBJECTIVE: To examine patient characteristics, persistence and adherence to treatment associated with use of second-generation antipsychotic long-acting injectable (SGA LAI) medications in the Australian real-world setting. METHOD: Five SGA LAIs were compared using a retrospective 10% sample of prescriptions in Australian Pharmaceutical Benefits Scheme (PBS) data: paliperidone palmitate 1-monthly (PP1M), paliperidone palmitate 3-monthly (PP3M), aripiprazole monohydrate (ARI), risperidone (RLAI) and olanzapine pamoate (OLAI). RESULTS: Patients in the PP3M cohort were more persistent with treatment (p < 0.001). Median months of persistence: PP3M (36 months); ARI (18 months); PP1M (11 months); OLAI (8 months); RLAI (4 months). Patients in the PP3M cohort were more adherent to treatment (p < 0.001): PP3M (78%); ARI (51%); PP1M (46%); OLAI (35%); RLAI (33%). CONCLUSIONS: Patients on PP3M treatment showed comparatively longer persistence and better adherence. Treatments for schizophrenia with longer dosing intervals may provide patients with symptomatic stability that could allow for reduced hospitalisations/relapse and increased focus on functional recovery.
Assuntos
Antipsicóticos , Esquizofrenia , Humanos , Esquizofrenia/tratamento farmacológico , Antipsicóticos/uso terapêutico , Palmitato de Paliperidona , Estudos Retrospectivos , AustráliaRESUMO
The pathological sequestration of TAR DNA-binding protein 43 (TDP-43, encoded by TARDBP) into cytoplasmic pathological inclusions characterizes the distinct clinical syndromes of amyotrophic lateral sclerosis and behavioural variant frontotemporal dementia, while also co-occurring in a proportion of patients with Alzheimer's disease, suggesting that the regional concentration of TDP-43 pathology has most relevance to specific clinical phenotypes. This has been reflected in the three different pathological staging schemes for TDP-43 pathology in these different clinical syndromes, with none of these staging schemes including a preclinical phase similar to that which has proven beneficial in other neurodegenerative diseases. To apply each of these three staging schemes for TDP-43 pathology, the clinical phenotype must be known undermining the potential predictive value of the pathological examination. The present study set out to test whether a more unified approach could accurately predict clinical phenotypes based solely on the regional presence and severity of TDP-43 pathology. The selection of brain regions of interest was based on key regions routinely sampled for neuropathological assessment under current consensus criteria that have also been used in the three TDP-43 staging schemes. The severity of TDP-43 pathology in these regions of interest was assessed in four clinicopathological phenotypes: amyotrophic lateral sclerosis (n = 27, 47-78 years, 15 males), behavioural variant frontotemporal dementia (n = 15, 49-82 years, seven males), Alzheimer's disease (n = 26, 51-90 years, 11 males) and cognitively normal elderly individuals (n = 17, 80-103 years, nine males). Our results demonstrate that the presence of TDP-43 in the hypoglossal nucleus discriminates patients with amyotrophic lateral sclerosis with an accuracy of 98%. The severity of TDP-43 deposited in the anterior cingulate cortex identifies patients with behavioural variant frontotemporal dementia with an accuracy of 99%. This identification of regional pathology associated with distinct clinical phenotypes suggests key regions on which probabilistic pathological criteria, similar to those currently available for Alzheimer's disease and dementia with Lewy bodies, can be developed for TDP-43 proteinopathies. We propose and validate a simplified probabilistic statement that involves grading the presence of TDP-43 in the hypoglossal nucleus and the severity of TDP-43 in the anterior cingulate for the pathological identification of TDP-43 proteinopathy cases with clinical amyotrophic lateral sclerosis and behavioural variant frontotemporal dementia.
Assuntos
Encéfalo/patologia , Proteínas de Ligação a DNA/metabolismo , Fenótipo , Proteinopatias TDP-43/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/patologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos TestesRESUMO
Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.
Assuntos
Dinaminas/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Hidrazonas/farmacologia , Naftóis/farmacologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Toxina da Cólera/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Dinaminas/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Naftóis/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Ovinos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Transferrinas/metabolismoRESUMO
Signalling from receptor tyrosine kinases is elicited by ligand binding which initiates the activation of many downstream signalling cascades. Endocytosis has been widely accepted as one mechanism in which cells inactivate signalling by internalising and subsequently degrading activated receptors. However, it is now evident that endocytosis of signalling receptors is important in initiation and sustaining downstream signalling. We and others have previously shown that epidermal growth factor receptor (EGFR) overexpression and activation of signal transducer and activator of transcription 3 (Stat3) are associated with tumourigenicity. Here, we examine the role of endocytosis in EGFR signal attenuation and differential signalling. Inhibition of dynamin II (Dyn II), a GTPase required for endocytosis, with a small molecular weight inhibitor, led to reduced EGF-mediated Stat3 phosphorylation and transcriptional activity in the A431 and HN5 human tumour cell lines. However, Dyn II inhibition had minimal effect on EGF-mediated EGFR and Erk1/2 phosphorylation, which is often regarded responsible for the tumourigenic function of the EGFR. Interestingly, this effect on Stat3 activation was not due to reduced EGFR/Stat3 association. Likewise, cells transfected with Dyn II siRNA or stably transfected with Dyn II shRNA had reduced EGF-mediated phospho-Stat3 levels but similar EGF-mediated phospho-EGFR and phospho-Erk1/2 levels compared with controls. Dyn II siRNA also reduced Stat3 transcriptional reporter activity and inhibits Stat3 accumulating into the nucleus. Taken together, our data suggest that the activation status of Stat3 and Erk1/2 and the sustainability of these signals are potentially due to the spatial and temporal control of the EGFR within the cell. This notion may have implications on therapeutic targeting and efficacy when using inhibitors to proteins either regulating endocytosis and/or signalling.
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Dinamina II/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular Tumoral , Inativação Gênica , Células HEK293 , Humanos , Camundongos , Microscopia Confocal/métodos , Modelos Biológicos , Fosforilação , Transdução de Sinais , Fatores de TempoRESUMO
The Bis-T series of compounds comprise some of the most potent inhibitors of dynamin GTPase activity yet reported, e. g., (2E,2'E)-N,N'-(propane-1,3-diyl)bis(2-cyano-3-(3,4-dihydroxyphenyl)acrylamide) (2), Bis-T-22. The catechol moieties are believed to limit cell permeability, rendering these compounds largely inactive in cells. To solve this problem, a prodrug strategy was envisaged and eight ester analogues were synthesised. The shortest and bulkiest esters (acetate and butyl/tert-butyl) were found to be insoluble under physiological conditions, whilst the remaining five were soluble and stable under these conditions. These five were analysed for plasma stability and half-lives ranged from â¼2.3â min (propionic ester 4), increasing with size and bulk, to greater than 24â hr (dimethyl carbamate 10). Similar profiles where observed with the rate of formation of Bis-T-22 with half-lives ranging from â¼25â mins (propionic ester 4). Propionic ester 4 was chosen to undergo further testing and was found to inhibit endocytosis in a dose-dependent manner with IC50 â¼8â µM, suggesting this compound is able to effectively cross the cell membrane where it is rapidly hydrolysed to the desired Bis-T-22 parent compound.
Assuntos
Pró-Fármacos , Pró-Fármacos/farmacologia , Dinaminas/farmacologia , Ésteres/farmacologia , EndocitoseRESUMO
This study proposes a practical approach, using the minimum number of brain regions and stains, to consolidate previously published neuropathological criteria into one operationalized schema to differentiate subtypes of frontotemporal lobar degeneration with tau-immunopositive inclusions (FTLD-tau). This approach uses the superior frontal and precentral cortices and hippocampus stained for phosphorylated-tau, p62 and modified Bielschowsky silver, and the midbrain stained only for modified Bielschowsky silver. Accuracy of interrater reliability was determined by 10 raters in 24 FTLD-tau cases (Pick disease = 4, corticobasal degeneration = 9, progressive supranuclear palsy = 5, globular glial tauopathy = 6) including 4 with a mutation in MAPT collected with consent by Sydney Brain Bank. All brain regions and stains assessed proved informative for accurate pathological subtyping, and many neuropathological features were identified as common across the FTLD-tau subtypes. By identifying subtype-specific neuropathological features in the sections selected, 10 independent observers assigned the cases to a FTLD-tau subtype with almost perfect agreement between raters, emphasizing the requirement for the assessment of subtype-specific features for the accurate subtyping of FTLD-tau. This study consolidates current consensus diagnostic criteria for classifying FTLD-tau subtypes with an efficient, simple and accurate approach that can be implemented in future clinicopathological studies.
Assuntos
Degeneração Lobar Frontotemporal/classificação , Degeneração Lobar Frontotemporal/patologia , Tauopatias/classificação , Tauopatias/patologia , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore.
Assuntos
Trifosfato de Adenosina/metabolismo , Espaço Extracelular/metabolismo , Complexos Multiproteicos/metabolismo , Miosinas/metabolismo , Receptores Purinérgicos P2/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Complexos Multiproteicos/química , Miosinas/química , Miosinas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X7RESUMO
INTRODUCTION: Exploring the degree of heritability in a large cohort of frontotemporal lobar degeneration with tau-immunopositive inclusions (FTLD-tau) and determining if different FTLD-tau subtypes are associated with stronger heritability will provide important insight into disease pathogenesis. METHODS: Using modified Goldman pedigree classifications, heritability was examined in pathologically proven FTLD-tau cases with dementia at any time (n = 124) from the Sydney-Cambridge collection. RESULTS: Thirteen percent of the FTLD-tau cohort have a suggested autosomal dominant pattern of inheritance, 25% have some family history, and 62% apparently sporadic. MAPT mutations were found in 9% of cases. Globular glial tauopathy was associated with the strongest heritability with 40% having a suggested autosomal dominant pattern of inheritance followed by corticobasal degeneration (19%), Pick's disease (8%), and progressive supranuclear palsy (6%). DISCUSSION: Similar to clinical frontotemporal dementia syndromes, heritability varies between pathological subtypes. Further identification of a genetic link in cases with strong heritability await discovery.
RESUMO
OBJECTIVE: To investigate the prevalence of clinically relevant multiple system atrophy (MSA) and Lewy body disease (LBD) pathologies in a large frontotemporal lobar degeneration (FTLD) cohort to determine if concomitant pathologies underlie the heterogeneity of clinical features. METHODS: All prospectively followed FTLD-tau and FTLD-TDP cases held by the Sydney Brain Bank (n = 126) were screened for coexisting MSA and LBD (Braak ≥ stage IV) pathology. Relevant clinical (including family history) and genetic associations were determined. RESULTS: MSA pathology was not identified in this series. Of the FTLD cohort, 9 cases had coexisting LBD ≥ Braak stage IV and were associated with different FTLD subtypes including Pick disease (n = 2), corticobasal degeneration (n = 2), progressive supranuclear palsy (n = 2), and TDP type A (n = 3). All FTLD-TDP cases with coexisting LBD had mutations in progranulin (n = 2) or an abnormal repeat expansion in C9orf72 (n = 1). All FTLD-tau cases with coexisting LBD were sporadic. The H1H1 MAPT haplotype was found in all cases that could be genotyped (n = 6 of 9). Seven cases presented with a predominant dementia disorder, 3 of which developed parkinsonism. Two cases presented with a movement disorder and developed dementia in their disease course. The age at symptom onset (62 ± 11 years) and disease duration (8 ± 5 years) in FTLD cases with coexisting LBD did not differ from pure FTLD or pure LBD cases in the brain bank. CONCLUSION: Coexisting LBD in FTLD comprises a small proportion of cases but has implications for clinical and neuropathologic diagnoses and the identification of biomarkers.
Assuntos
Degeneração Lobar Frontotemporal/epidemiologia , Doença por Corpos de Lewy/epidemiologia , Atrofia de Múltiplos Sistemas/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Proteína C9orf72/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Degeneração Lobar Frontotemporal/fisiopatologia , Humanos , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/patologia , Doença por Corpos de Lewy/fisiopatologia , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Prevalência , Progranulinas/genética , alfa-Sinucleína/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 +/- 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.
Assuntos
Alcanos/farmacologia , Dinamina II/antagonistas & inibidores , Dinamina I/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Compostos de Amônio Quaternário/farmacologia , Compostos de Trimetil Amônio/farmacologia , Alcanos/química , Animais , Células COS , Chlorocebus aethiops , Dinamina I/fisiologia , Dinamina II/fisiologia , Células HeLa , Humanos , Compostos de Amônio Quaternário/química , Ovinos , Tensoativos/química , Tensoativos/farmacologia , Compostos de Trimetil Amônio/químicaRESUMO
Dynamin I is a GTPase enzyme required for endocytosis and is an excellent target for the design of potential endocytosis inhibitors. Screening of a library of tyrphostins, in our laboratory, against the GTPase activity of dynamin I gave rise to a microM potent lead, 2-cyano-3-(3,4-dihydroxyphenyl)thioacrylamide (1, IC50 70 microM). Our initial investigations suggested that only the dimeric form of 1 displayed dynamin I GTPase inhibitory activity. Subsequent synthetic iterations were based on dimeric analogues and afforded a number of small molecules, low microM potent, inhibitors of dynamin I GTPase, in particular, symmetrical analogues with a minimum of two free phenolic -OHs: catechol-acrylamide (9) (IC50= 5.1 +/- 0.6 microM), its 3,4,5-trihydroxy congener (10) (IC50= 1.7 +/- 0.2 microM), and the corresponding 3-methyl ether (11) (IC50= 9 +/- 3 microM). Increasing the length of the central alkyl spacer from ethyl to propyl (22-24) afforded essentially identical activity with IC50's of 1.7 +/- 0.2, 1.7 +/- 0.2, and 5 +/- 1 microM, respectively. No decrease in activity was noted until the introduction of a hexyl spacer. Our studies highlight the requirement for two free amido NHs with neither the mono-N-methyl (86) nor the bis-N-methyl (87) analogues inhibiting dynamin I GTPase. A similar effect was noted for the removal of the nitrile moieties. However, modest potency was observed with the corresponding ester analogues of 9-11: ethyl ester (90), propyl ester (91), and butyl ester (92), with IC50's of 42 +/- 3, 38 +/- 2, and 61 +/- 2 microM, respectively. Our studies reveal the most potent and promising dynamin I GTPase inhibitor in this series as (22), which is also known as BisT.
Assuntos
Dinamina I/antagonistas & inibidores , Tirfostinas/síntese química , Animais , Encéfalo/enzimologia , Dimerização , Dinamina I/química , Ovinos , Relação Estrutura-Atividade , Tirfostinas/químicaRESUMO
BACKGROUND: Two commercially available TDP43 antibodies (phosphorylated or pTDP43, non-phosphorylated or iTDP43) are currently in use for the neuropathological classification of FTLD-TDP cases into pathological subtypes. To date, no studies have performed direct comparisons between these TDP43 antibodies to determine if they identify the same FTLD-TDP subtypes. The reliability of subtype classification with the use of either of these antibodies has also not been investigated. The present study compares the severity of pathological lesions identified with pTDP43 and iTDP43 in a cohort of 14 FTLD-TDP cases, and assesses the accuracy and inter-observer reliability found with either of these antibodies. RESULTS: pTDP43 identified a greater severity of pathological inclusions across FTLD-TDP cases in comparison to iTDP43 and a higher inter-observer of subtype classification was found with this antibody. CONCLUSION: This study demonstrates a higher consistency across independent observers in the pathological subtyping of FTLD-TDP cases with the use of a pTDP43 antibody in comparison to the iTDP43 antibody, and corroborates the use of pTDP43 for pathological classification of FTLD-TDP cases.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/classificação , Degeneração Lobar Frontotemporal/metabolismo , Imuno-Histoquímica/métodos , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Estudos de Coortes , Feminino , Degeneração Lobar Frontotemporal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Reprodutibilidade dos Testes , Índice de Gravidade de DoençaRESUMO
Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 µM for dynamin I and 1.8 µM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin's role in endocytosis.
Assuntos
Desenho de Fármacos , Dinaminas/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Bioensaio , Western Blotting , Células COS , Chlorocebus aethiops , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Hebbian synaptic plasticity, such as hippocampal long-term potentiation (LTP), is thought to be important for particular types of learning and memory. It involves changes in the expression and activity of a large array of proteins, including cell adhesion molecules. The integrin class of cell adhesion molecules has been extensively studied in this respect, and appear to have a defined role in consolidating both structural and functional changes brought about by LTP. With the use of integrin inhibitors, it has been possible to identify a critical time window of several minutes after LTP induction for the participation of integrins in LTP. Altering the interactions of integrins with their ligands during this time compromises structural changes involving actin polymerisation and spine enlargement that could be required for accommodating new AMPA receptors (AMPARs). After this critical window of structural remodelling and plasticity, integrins "lock-in" and stabilise the morphological changes, conferring the requisite longevity for LTP. Genetic manipulations targeting integrin subtypes have helped identify the specific integrin subunits involved in LTP and correlate alterations in plasticity with behavioural deficits. Moreover, recent studies have implicated integrins in AMPAR trafficking and glycine receptor lateral diffusion, highlighting their multifaceted functions at the synapse.
Assuntos
Integrinas/fisiologia , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Humanos , Sinapses/ultraestruturaRESUMO
Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.
Assuntos
Benzopiranos/síntese química , Dinamina II/antagonistas & inibidores , Dinamina I/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Animais , Benzopiranos/química , Benzopiranos/farmacologia , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Cinética , Masculino , Ratos , Ratos Sprague-Dawley , Receptores da Transferrina/metabolismo , Ovinos , Relação Estrutura-Atividade , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM; RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.
Assuntos
Acrilamidas/síntese química , Acrilamidas/farmacologia , Dinamina I/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/farmacologia , Acrilamidas/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Dinamina I/metabolismo , Inibidores Enzimáticos/química , Humanos , Indóis/química , Camundongos , Estrutura Molecular , Células NIH 3T3 , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The survival of motor neurons is controlled by multiple factors that regulate different aspects of their physiology. The identification of these factors is important because of their relationship to motor neuron disease. We investigate here whether Mullerian Inhibiting Substance (MIS) is a motor neuron survival factor. We find that motor neurons from adult mice synthesize MIS and express its receptors, suggesting that mature motor neurons use MIS in an autocrine fashion or as a way to communicate with each other. MIS was observed to support the survival and differentiation of embryonic motor neurons in vitro. During development, male-specific MIS may have a hormone effect because the blood-brain barrier has yet to form, raising the possibility that MIS participates in generating sex-specific differences in motor neurons.