Preclinical cardiovascular safety assessment of pharmacology-toxicology relationship for a set of novel kinase inhibitors.

Cardiovascular toxicity is one of the more common causes of attrition in preclinical and clinical drug development. Preclinical cardiovascular safety assessment involves numerous in vitro and in vivo endpoints which are being continually reviewed and improved to lower the incidence of cardiovascular toxicity that manifests only after the initiation of clinical trials. An example of notable preclinical toxicity is necrosis in the papillary muscle of the left ventricle in dogs that is induced by exaggerated pharmacological effects of vasodilators or positive inotropic/vasodilating off-target drug effects. Two distinct, small-molecule inhibitors that target an intracellular kinase, Compound A and Compound B, were profiled in 2-week dose-range finding and 4-week toxicity studies. Serum cardiac troponin (cTnI) was evaluated after a single dose and after 2-week and 4-week repeat dose studies with each kinase inhibitor. Acute effects on hemodynamic (heart rate, blood pressures, left ventricular contractility) and electrocardiographic (QTcV, PR, QRS intervals) endpoints by each inhibitor were assessed in an anesthetized dog cardiovascular model. Cardiovascular degeneration/necrosis with and without fibrosis was observed in dogs and correlated to increases in serum cTnI in repeat-dose toxicity studies. At the same doses used in toxicologic assessments, both kinase inhibitors produced sustained increases in heart rate, left ventricular contractility, and cardiac output, and decreases in mean arterial pressure. Cardiac pathology findings associated with these 2 kinase inhibitors were accompanied not only by cardiac troponin elevations but also associated with hemodynamic changes, highlighting the importance of the link of the physiologic-toxicologic interplay in cardiovascular safety assessment.

Upregulation of PCSK9, rho kinase and cardiac troponin by Eucalyptus globulus leaf extract improves fructose-streptozotocin-induced diabetic cardiac dysfunction in rats.

CONTEXT: The effect of Eucalyptus globulus in diabetic cardiac dysfunction and the possible mechanisms involved have not been explored. OBJECTIVE: To evaluate the effect of ethanol leaf extract of E. globulus (NEE) on the cardiac function of fructose/streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Type-2 diabetes was induced in rats with 10% fructose feeding for 14 days and an intraperitoneal administration of 40 mg/kg streptozotocin. Diabetic animals were treated with NEE (100-400 mg/kg) or 5 mg/kg glibenclamide orally for 21 days. Biochemical assays, histopathological examination and analyses of PCSK9, Rho kinase and Cardiac troponin expression were performed. RESULTS: The untreated diabetic group showed decreased expression of the genes, oxidative stress, dyslipidemia, increased activities of creatine kinase MB and lactate dehydrogenase, reduced nitric oxide level, and depletion of cardiomyocytes, which were reversed in NEE treated groups. CONCLUSIONS: Eucalyptus globulus ameliorated diabetic cardiac dysfunction through increased PCSK9, Rho kinase and Cardiac troponin expression.

ESMOLOL PROTECTS AGAINST LPS-INDUCED CARDIAC INJURY VIA THE AMPK/mTOR/ULK1 PATHWAY IN RAT.

ABSTRACT: Aim: The purpose of this study was to investigate the effect of esmolol (ES) on LPS-induced cardiac injury and the possible mechanism. Methods: Sepsis was induced by i.p. injection of LPS (10 mg/kg) in male Sprague-Dawley rats pretreated with ES, 3-methyladenine or rapamycin. The severity of myocardial damage was analyzed by hematoxylin-eosin staining, and myocardial damage scores were calculated. The concentration of cardiac troponin was measured by enzyme-linked immunosorbent assay. The expression of autophagy-related proteins (beclin-1, LC3-II, p-AMPK, p-ULK1, p-mTOR) in myocardial tissue was detected by Western blotting. Autophagosome formation and the ultrastructural damage of mitochondria were assessed using transmission electron microscopy. Results: LPS induced an increase in myocardial damage score in a time-dependent manner, accompanied with an increase in autophagy at 3 h and decrease in autophagy at 6, 12, and 24 h. Pretreatment of LPS-treated rats with ES or rapamycin reduced myocardial injury (release of cardiac troponin, myocardial damage score) and increased autophagy (LC3-II, beclin-1, p-AMPK, and p-ULK1 levels and autophagosome numbers) at 12 and 24 h. In contrast, 3-methyladenine showed no effect. Conclusion: Esmolol alleviates LPS-induced myocardial damage through activating the AMPK/mTOR/ULK1 signal pathway-regulated autophagy.

Modern Concepts of the Role of Fine Particles (PM 2.5) in the Genesis of Atherosclerosis and Myocardial Damage: Clinical and Epidemiological Data, the Main Pathophysiological Mechanisms.

Due to the fact that atherosclerotic cardiovascular diseases (CVDs) dominate in the structure of morbidity, disability and mortality of the population, the study of the risk factors for the development of atherosclerotic CVDs, as well as the study of the underlying pathogenetic mechanisms thereof, is the most important area of scientific research in modern medicine. Understanding these aspects will allow improving the set of treatment and preventive measures and activities. One of the important risk factors for the development of atherosclerosis, which has been actively studied recently, is air pollution with fine particulate matter (PM 2.5). According to clinical and epidemiological data, the level of air pollution with PM 2.5 exceeds the normative indicators in most regions of the world and is associated with subclinical markers of atherosclerosis and mortality from atherosclerotic CVDs. The aim of this article is to systematize and discuss in detail the role of PM 2.5 in the development of atherosclerosis and myocardial damage with the consideration of epidemiological and pathogenetic aspects. Materials and Methods: This narrative review is based on the analysis of publications in the Medline, PubMed, and Embase databases. The terms "fine particles" and "PM 2.5" in combination with "pathophysiological mechanisms," "cardiovascular diseases", "atherosclerosis", "cardiac troponins", "myocardial damage" and "myocardial injury" were used to search publications. Conclusion: According to the conducted narrative review, PM 2.5 should be regarded as the significant risk factor for the development of atherosclerotic CVDs. The pro-atherogenic effect of fine particulate matter is based on several fundamental and closely interrelated pathophysiological mechanisms: endothelial dysfunction, impaired lipid metabolism, increased oxidative stress and inflammatory reactions, impaired functioning of the vegetative nervous system and increased activity of the hemostatic system. In addition, PM 2.5 causes subclinical damage to cardiac muscle cells by several mechanisms: apoptosis, oxidative stress, decreased oxygen delivery due to coronary atherosclerosis and ischemic damage of cardiomyocytes. Highly sensitive cardiac troponins are promising markers for detecting subclinical myocardial damage in people living in polluted regions.

Relaxin-3 Ameliorates Diabetic Cardiomyopathy by Inhibiting Endoplasmic Reticulum Stress.

Background: This study is aimed at investigating whether relaxin-3 exhibits protective effects against cardiomyopathy in diabetic rats by suppressing ERS. Methods: Eighty male SD rats were randomly divided into two groups: controls (n = 20) and diabetes (n = 60). The streptozotocin-treated rats were randomly divided into three groups: diabetic group (DM), low-dose relaxin-3 group (0.2 µg/kg/d), and high-dose relaxin-3 group (2 µg/kg/d). The myocardial tissues and collagen fiber were observed by hematoxylin and eosin (H&E) and Masson staining. Serum brain natriuretic peptide (BNP), troponin (TNI), myoglobin, interleukin (IL-17), interleukin (IL)-1α, and tumor necrosis factor (TNF)-α were determined by ELISA. The protein expression of glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the heart tissue of each group was detected by Western blot analysis. Results: (1) HE and Masson staining indicated that relaxin-3 could attenuate myocardial lesions and myocardial collagen volume fraction. (2) BNP, TnI, and myoglobin in the DM group at four and eight weeks were significantly higher than in the controls (P < 0.01). The relaxin-3-treated groups showed significantly reduced serum BNP, TnI, and myoglobin levels compared with the DM group (P < 0.05). (3) IL-17, IL-1α, and TNF-α levels in the DM rats at 4 weeks were higher than in the controls (P < 0.05). Low or high dose of relaxin-3-treated groups showed reduced serum IL-17 and TNF-α levels compared with the DM group at four and eight weeks (P < 0.05). (4) CHOP and GRP78 protein expression was increased in the DM group at four and eight weeks compared with the controls (P < 0.01), and small and large doses of relaxin-3 significantly reduced GRP78 and CHOP protein expression. Conclusions: Exogenous relaxin-3 ameliorates diabetic cardiomyopathy by inhibiting ERS in diabetic rats.

Protective Effect of Curcumin, Chrysin and Thymoquinone Injection on Trastuzumab-Induced Cardiotoxicity via Mitochondrial Protection.

Mitochondrial dysfunction may lead to cardiomyocyte death in trastuzumab (TZM)-induced cardiotoxicity. Accordingly, this study was designed to evaluate the mitochondrial protective effects of curcumin, chrysin and thymoquinone alone in TZM-induced cardiotoxicity in the rats. Forty-eight male adult Wistar rats were divided into eight groups: control group (normal saline), TZM group (2.5 mg/kg I.P. injection, daily), TZM + curcumin group (10 mg/kg, I.P. injection, daily), TZM + chrysin (10 mg/kg, I.P. injection, daily), TZM + thymoquinone (0.5 mg/kg, I.P. injection, daily), curcumin group (10 mg/kg, I.P. injection, daily), chrysin group (10 mg/kg, I.P. injection, daily) and thymoquinone group (10 mg/kg, I.P. injection, daily). Blood and tissue were collected on day 11 and used for assessment of creatine phosphokinase, lactate dehydrogenase (LDH), troponin, malondialdehyde (MDA) amount, glutathione levels and mitochondrial toxicity parameters. TZM increased mitochondrial impairments (reactive oxygen species formation, mitochondrial swelling, mitochondrial membrane potential collapse and decline in succinate dehydrogenase activity) and histopathological alterations (hypertrophy, enlarged cell, disarrangement, myocytes degeneration, infiltration of fat in some areas, hemorrhage and focal vascular thrombosis) in rat heart. As well as TZM produced a significant increase in the level of CK, LDH, troponin, MDA, glutathione disulfide. In most experiments, the co-injection of curcumin, chrysin and thymoquinone with TZM restored the level of CK, LDH, troponin, MDA, GSH, mitochondrial impairments and histopathological alterations. The study revealed the cardioprotective effects of curcumin, chrysin and thymoquinone against TZM-induced cardiotoxicity which could be attributed to their antioxidant and mitochondrial protection activities.

Skin wound healing with composite biomembranes loaded by tiopronin or captopril.

Novel wound dressings composed of chitosan (Ch) and hyaluronan (HA) loaded with tiopronin or captopril as antiinflammatory drugs were prepared. Composite biomembranes were examined in skin wounds of ischemic rabbits with the aim to accelerate the process of healing. The results proved that the biomembranes composed of Ch/HA/tiopronin or Ch/HA/captopril facilitated healing of skin wounds compared to untreated animals and animals treated with Ch/HA membranes. These results were confirmed by histology. Cu(II) ions and ascorbate-induced high-molar-mass HA degradation by means of rotational viscometry was performed and the ability of the both drugs to scavenge reactive oxygen species was evaluated. The results showed that captopril as well as tiopronin decreased the rate of HA degradation exclusively at higher concentrations.

Monitoramento do horizonte tecnológico: Medicamentos em desenvolvimento para o tratamento da atrofia muscular espinhal (AME) / Monitoring the technological horizon: Drugs under development for the treatment of spinal muscular atrophy (SMA)

A DOENÇA: As Atrofias Musculares Espinhais (AME) são um conjunto de doenças neuromusculares de etiologia genética relacionadas à hereditariedade de genes autossômicos recessivos. Os sinais e sintomas da AME são provenientes da degeneração e perda dos neurônios motores na medula espinhal e tronco encefálico, resultando em fraqueza muscular progressiva. Essas atrofias apresentam grande variabilidade clínica ocasionada pela perda ou deficiência da proteína de sobrevivência do neurônio motor (SMN - do inglês: Survival Motor Neuron). A maioria dos casos (95-98%) ocorre pela deleção homozigótica do gene SMN1, porém também pode ser decorrente de mutação heterozigótica pontual em um dos alelos desse gene. A mutação do gene SMN1 no cromossomo não sexual de número cinco, específica no loci denominado 5q11-13, dá origem à AME proximal ou denominada 5q. Essa mutação leva a uma troca de uma base nitrogenada citosina para timina, ocasionando a deleção do éxon 7. Dessa forma, essa mutação resulta em um RNA mensageiro (RNAm) encurtado, que codifica proteínas SMN truncadas, em menor quantidade e mais instáveis, sendo rapidamente degradadas. Um outro gene, o SMN2, também produz proteínas SMN, similares às transcritas pelo gene SMN1, contudo em uma quantidade menor. Assim, a presença do gene SMN2 em pacientes com AME mimetizam as manifestações clínicas da doença, ou seja, funciona como uma reserva de segurança para a perda do gene SMN. TRATAMENTO: Atualmente, o tratamento farmacológico da AME é limitado. Três medicamentos possuem registros de comercialização no mundo com indicação em bula para a doença. São eles: nusinersena, onasemnogeno abeparvoveque e ridisplam, este último registrado pelo Food and Drug Administration (FDA) em agosto de 2020. Os dois primeiros já estão registrados também no Brasil. O nusinersena é um medicamento administrado por via intratecal que atua como um oligonucleotídeo. antissenso (ou antissentido). Por meio de uma ligação à região intrônica 7, o nusinersena impede que os fatores de silenciamento/remoção intrônicos processem e removam o éxon 7 do RNAm do gene SMN2. A retenção do éxon 7 no RNAm de SMN2, permite a leitura e tradução correta destes genes, potencializando a produção de proteína funcional relacionada com a sobrevivência do neurônio motor, a proteína SMN (13). O medicamento foi registrado pelo FDA em 2016 e pela Anvisa em 2017. ESTRATÉGIA DE BUSCA: Para localizar os medicamentos em fase de pesquisa clínica para AME, foram consultados os sítios eletrônicos ClinicalTrials. gov, Embase, Medline (via PubMed) e no Google Acadêmico utilizando os termos "Spinal Muscular Atrophy". As buscas foram realizadas em 17 de julho de 2019. Consideraram-se as tecnologias a partir da fase II de pesquisa clínica, com a AME como alvo e sem registro para essa indicação terapêutica no Brasil. Posteriormente, utilizando-se os códigos de registro do ClinicalTrials.gov referentes aos estudos clínicos com os medicamentos identificados na etapa descrita no parágrafo anterior e aos nomes de cada uma dessas tecnologias, realizou-se busca nas bases de dados MEDLINE (via PubMed) e EMBASE, para a pesquisa de resultados publicados dos estudos clínicos. Além disso, uma busca complementar por resultados desses estudos publicados em anais de congressos científicos também foi realizada. MEDICAMENTOS EM FASE DE PESQUISA CLÍNICA: Modificador do padrão de splicing. Firefish. Sunfish. Jewelfish. Firefish + Sunfish + Jewelfish. Branaplan. Terapia Gênica: Onasemnogeno Abeparvoveque. Modificadores da Contração Muscular: Piridostigmina. CK-212707/ CK-107. SRK-015. Amifampridina. Anti-Inflamatório não Esteroidal que Altera os Níveis de Proteína SMN: Celecoxibe. LIMITAÇÕES: A maioria dos estudos localizados ainda está em andamento, estando disponíveis apenas resultados de análises interinas ou parciais apresentados em eventos científicos. Deste modo, as informações estão disponíveis apenas por meio de resumos ou pôsteres e alguns dados relevantes não estão disponíveis. No estudo FIREFISH, foi relatado que mais pacientes em uso de risdiplam não tiveram complicações decorrentes da doença, mas não informa quantos ou qual a proporção de indivíduos que apresentaram este resultado (19­22). Já a respeito do estudo SUNFISH, os autores reportaram a ocorrência de eventos adversos leves, mas não informaram a natureza e tipo destes eventos. INFORMAÇÕES ADICIONAIS: O Institute for Clinical and Economical Review (ICER) publicou relatório comparando o onasemnogeno abeparvoque ao nusinersena em termos clínicos e econômicos. No caso do nusinersena, os benefícios são mais evidentes entre pacientes pré-sintomáticos. Ambas as terapias possuem custo elevado e as análises de custo-efetividade do nusinersena mostram valores de custo-efetividade incremental superiores aos comumente aceitos nos Estados Unidos, tanto para anos de vida ajustados à qualidade (AVAQ) quanto para anos de vida ganhos (AVG). Quanto ao onasemnogeno abeparvoveque, no momento da avaliação ainda não havia sido definido um preço para o medicamento, embora os autores acreditem que ele também apresentará custo elevado e razão de custo efetividade incremental, seja por anos de vida ajustados à saúde, ou por anos de vida ganho superior aos limiares de custo-efetividade comumente utilizados. A conclusão dos autores do relatório foi de que ambas as terapias poderiam trazer benefícios clínicos importantes ao paciente, em termos de mortalidade, necessidade de ventilação mecânica e marcos de desenvolvimento motor para pacientes com AME tipo I. Ao obter aprovação do FDA, o onasemnogeno abeparvoveque teve seu preço estipulado em 2,1 milhões de dólares. A empresa fabricante do medicamento informou que irá trabalhar com os pagadores para implementar acordos de 5 anos baseados em desfechos e em opções de pagamentos ao longo do tempo. Estas informações, embora sejam provenientes de um cenário diferente do brasileiro, podem trazer questões relevantes para tomadas de decisão acerca de incorporações. Embora os Estados Unidos e o Brasil apresentem diferenças quanto ao cenário econômico e o tipo de sistema de saúde, problemas relacionados a restrições orçamentárias ocorrem em ambas as nações. Em termos de custo-efetividade, embora não tenham sido realizadas análises econômicas, é provável que ambos os medicamentos apresentem razões de custo-efetividade incremental elevadas. CONCLUSÕES: A AME é uma doença altamente incapacitante e é a causa mais comum de mortalidade infantil dentre as condições hereditárias. Sua heterogeneidade clínica resulta em dificuldades para o desenvolvimento de terapias efetivas e de estudos clínicos adequados. O objetivo do tratamento é promover melhoras na função motora e retardar a progressão de doença. Nos últimos dois anos, a perspectiva de tratamento medicamentoso da AME apresentou um avanço. O medicamento nusinersena possui registro no Brasil e foi incorporado ao SUS no primeiro semestre de 2019 para o tratamento de AME 5q tipo I. O onasemnogeno abeparvoveque foi registrado em agosto de 2020 para uso no Brasil, indicado para pacientes com AME com menos de dois anos de idade, e ainda carece de avaliação para incorporação no SUS (Informação atualizada em 24/09/ 2020). Por sua vez, o risdiplam foi registrado no FDA em agosto de 2020. A principal vantagem do risdiplam se deve ao fato da sua administração ser por via oral, porém o medicamento apresenta uso contínuo. A comparação publicada em 2019 pelo ICER quanto aos as pectos clínicos e econômicos do nusinersena e do onasemnogeno abeparvoveque conclui que ambas terapias trazem benefícios clínicos importantes para pacientes com AME 5q tipo I, sendo os efeitos do nusinersena mais evidentes em pacientes com AME pré-sintomática. As análises econômicas mostraram que as razões de custo-efetividade do nusinersena superam os limiares comumente aceitos nos Estados Unidos para AVAQ e AVG. Embora o onasemnogeno abeparvoveque não tivesse preço definido no momento da análise, é esperado que a mesma tendência seja observada. Foram localizadas ainda seis tecnologias em investigação (branaplan, piridostigmina, reldesemtiv, SRK-15, amifampridina, celecoxibe), entretanto, ainda em fases iniciais de estudo, com pouca ou nenhuma informação sobre seus resultados. Além disso, o perfil de segurança e eficácia dessas tecnologias precisa ser confirmado por meio de mais estudos clínicos comparativos. Desta forma, verifica-se que há no horizonte tecnológico potenciais tecnologias em desenvolvimento e que futuramente poderão ser utilizadas no tratamento da AME.

Sesgo en la estratificación del síndrome coronario agudo al calcular el grace- score con 1ª, 2ª o troponina Δ / Grace score bias in acute coronary syndrome due to use 1th, 2th or troponin Δ

Introducción: el síndrome coronario agudo (SCA) es la primera causa de mortalidad en Colombia. Una estratificación de riesgo errónea, en la sala de emergencias (ER), afecta las intervenciones realizadas y la tasa de eventos adversos cardiovasculares puede ser mayor. El objetivo de esta investigación fue medir la diferencia en el puntaje GRACE y la estratificación del riesgo coronario, utilizando los resultados de las troponinas medidas secuencialmente durante la atención inicial. Metodología: con un diseño descriptivo retrospectivo, se evaluaron los registros clínicos de pacientes tratados por dolor precordial de probabilidad intermedia para SCA, sin indicación de manejo invasivo inmediato, atendidos en la sala de emergencias de una clínica del tercer nivel de Bogotá, durante el año 2017. Se determinó la diferencia entre la puntuación GRACE calculada con la primera troponina (GRACE-1), la segunda troponina (GRACE-2) o la troponina delta (GRACE-delta) [prueba T pareada], y la proporción de pacientes poco estratificados se midió al usar la primera troponina [X2, puntaje Z]. Resultados: se identificaron 44 pacientes en un período de 6 meses. La mayoría hombres con edad mediana de 73 años. El promedio (DE) de los puntajes GRACE-1, GRACE-2 y GRACE-delta, fue de 114.14 (30.73), 115.55 (30.14) y 111.11 (28.79), respectivamente; al comparar GRACE-delta con GRACE-1 y GRACE-2 se identificaron diferencias significativas (p:<0.05). Se identificó un error en la estratificación del riesgo coronario en 10/44 pacientes (22.7%) y 9/44 (20.4%) presentaron sobreestratificación. Conclusión: la estratificación del riesgo coronario con la primera troponina, a diferencia de la troponina delta (ítem no aclarado en las guías), evidenció una sobreestratificación en al menos 20% de los pacientes, estableciendo la necesidad de procedimientos más invasivos y posiblemente hospitalización más prolongada permanecer.

Background: Acute coronary syndrome (ACS) is the first cause of mortality in Colombia. An erroneous risk stratification, in the emergency room (ER), affects the interventions performed and the rate of major cardiovascular adverse events. We measured the difference in GRACE score and stratification of coronary risk, by using the results of troponins measured sequentially during initial care. Methods: With a retrospective descriptive design, clinical records of patients treated for precordial pain of ≥ intermediate probability for ACS were evaluated, without indication of immediate invasive management, attended in the ER of a clinic of the third level of Bogotá, during 2017. De-termined the difference between the GRACE score calculated with the first (GRACE-1), second (GRACE-2) or troponin delta (GRACE-delta [paired T-test], and the proportion of poorly stratified patients was measured when using the first troponin [X2, Z-score]. Results: 44 patients in a period of 6 months were identified. The majority men, older adults, middle age 73 years. The average (SD) of scores GRACE-1, GRACE-2 and GRACE-delta, was 114.14 (30.73), 115.55 (30.14) and 111.11 (28.79), respectively; when comparing GRACE-delta with GRACE-1 and GRACE-2 significant differences were identified (p:<0.05). Error in the stratification of coronary risk was identified in 10/44 patients (22.7%), and 9/44 (20.4%) presented over-stratification. Conclusion: The stratification of coronary risk using the first troponin, unlike the troponin delta (item not clarified in the guidelines), evidenced an over-stratification in at least 20% of the patients, establishing the need for more invasive procedures and possibly longer hospital stay.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.

Plant troponoids: chemistry, biological activity, and biosynthesis.

Tropone or tropolone and its derivatives (here together called troponoids) belong to a family of natural products with a seven-membered aromatic ring and various side groups. They are mainly synthesized by plants and fungi, and most troponoids play roles of antibacterial defenses in these organisms. With an increasingly severe situation of antibiotic resistant bacteria, as well as a requirement for antifungal medicines, troponoids have attracted extensive studies since they have powerful antibacterial and antifungal activity, particularly against antibiotic-resistant bacteria. In addition, many other biological activities such as antiviral, antitumor, antioxidant, anti-inflammatory, insecticidal, or enzyme inhibitor activities are associated with troponoids. After extensive studies in the 1960s-70s, interests in natural troponoids dedclined. However, chemical and biomedical studies on troponoids bloom again from the 1990s. To date great progress has been made with troponoid study in terms of identification of new natural troponoids, chemical synthesis and properties, biological activity, biosynthesis and metabolism. Particularly, bioassay-guided screening strategy and structure-activity relation-directed structure modification and drug design has resulted in the synthesis and discovery of many new derivatives. Many of them have great promise to be developed into new medicines for their potent and specific activities. This review presents the recent advances in troponoid studies and highlights multiple faceted biological activities of troponoids, as well as their relationships with chemical structures. Chemistry, biosynthesis, and production via biotechnology of troponoids are also briefly reviewed. Applications of troponoids in daily life, agriculture, medicine, and industry, and the related patents have been considered to further extend our understanding of the increasing impact of troponoids on humans.

Effects of troponin on thermal unfolding of actin-bound tropomyosin.

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8 degrees C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48 degrees C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70 degrees C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100 degrees C. However, addition of TnI, and especially of TnT, to the F-actin-Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae.

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition.

Cooperative inhibition of actin filaments in the absence of tropomyosin.

We measured the inhibition of actin activated myosin subfragment-1 MgATPase activity in a solution containing no added KCl (5 mM PIPES.K2 (pH 7.1), 2.5 mM MgCl2, 1 mM DTT, 1 mM NaN3, 5 mM MgATP). Maximal inhibition was observed with substoichiometric concentrations of caldesmon, caldesmon domain 4, troponin and troponin I. In six experiments using different preparations of actin, S-1 and caldesmon 50% inhibition required 0.09 +/- 0.01 (sem) caldesmon added per actin. This compares with 0.66 +/- 0.32 (sem, n = 5) caldesmon per actin for 50% inhibition in the presence of 60 mM KCl. With caldesmon domain 4, 50% inhibition was achieved with 0.17 +/- 0.08 (n = 11) domain 4 added per actin. We measured the amount of caldesmon bound at the same time as inhibition. Complete inhibition of actin activated ATPase needed only one caldesmon bound per 5.0 +/- 0.5 (sem, n = 5) actin monomers or one caldesmon domain 4 bound per 3.9 +/- 0.6 (sem, n = 3) actin monomers at zero KCl. We conclude that under these conditions inhibition of actin is cooperative despite the absence of tropomyosin. We measured the effect of caldesmon inhibition upon S-1 binding to actin. S-1.ADP.Pi (weak binding) was not affected by caldesmon concentrations giving 80% inhibition, however S-1.ADP (strong binding) was highly cooperative, being very weak at <0.3 microM but indistinguishable from uninhibited actin at >2 microM S-1.ADP. We conclude that actin can exist in two activity states corresponding to the 'on' and 'off' states of actin-tropomyosin and inhibitory proteins function as allosteric-cooperative inhibitors of actin. The implications of these findings for the role of tropomyosin in thin filament regulation are discussed.

Fluorescence changes on contractile activation in TnC(DANZ) labeled skinned rabbit psoas fibers.

The increase in fluorescence of dansylaziridine (DANZ) labeled troponin C (TnC(DANZ) substituted into skinned rabbit psoas fibers was determined as a function of the pCa. The fluorescence data are expressed as the ratio of two wavelength bands, one that sees the fluorescence of TnC(DANZ), and one that sees background fluorescence and scatter. The percent TnC replaced with TnC(DANZ) was varied between 10 and 50% and, the fibers were randomly stretched, at the start of each experiment, between 10 and 50%. A large ratio increase accompanies increase in [Ca2+]. The pCa/force data are best fit by the Hill equation but the pCa/ratio data are best fit by a model in which Ca2+ binds in two phases. The position of the force curve on the pCa axis varies little between fibers, in contrast to that of the ratio or A-fluorescence curve. In accord with previous reports the delta-fluorescence can be left of the force on the pCa axis (type I) or superimpose in part on the force (type II). Not described previously, we find curves in which the second phase of the ratio cross-over the pCa/force curve. This type III relationship is found only in fibers less than 3 weeks postmuscle harvest. We propose that the first, relatively invariant, phase of the biphasic pCa/ratio curve accompanies Ca2+ binding to either of the two low affinity sites on TnC(DANZ) as it does for TnC in solution. The second, highly cooperative, phase of the ratio curve that accompanies muscle contraction and enhanced Ca2+ binding is initiated when sufficient Ca2+ is bound to overcome inhibitory systems. Loose coupling between the initial Ca2+ binding and the cooperative switch point may account for much of the variation in the shape and position of the pCa/ratio curve. There is evidence that, in the overlap zone, weakly attached myosin cross-bridges enhance cooperation between the regulatory units of the thin filaments.

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.

Powerful activation of skeletal muscle actomyosin ATPase by goniodomin A is highly sensitive to troponin/tropomyosin complex.

Goniodomin A has been shown to cause the conformational change of actin to modify actomyosin ATPase activity. Goniodomin A induced a potent stimulation of the actomyosin ATPase activities of the actin-myosin reconstituted system and natural actomyosin in the range of 10(-8) to 10(-7) M. When the concentration was increased above 10(-7) M, actomyosin ATPase activity was decreased. Interestingly, the troponin/tropomyosin complex caused a concentration-dependent inhibition of the goniodomin A-induced stimulation of actomyosin ATPase activity. In the presence of a high concentration of the troponin/tropomyosin complex, goniodomin A decreased actomyosin ATPase activity in a concentration-dependent manner. The enhancement of the ATPase activity of troponin/tropomyosin-free natural actomyosin by goniodomin A was larger than that obtained with natural actomyosin. Goniodomin A at lower concentrations enhanced the superprecipitation of natural actomyosin but decreased it at higher concentrations. The ATPase activity of skeletal muscle myofibrils and the contractile response of skinned fibers to Ca(2+) were never activated and were decreased by this compound, suggesting an inhibition by the troponin/tropomyosin complex. In the far ultraviolet circular dichroism, goniodomin A above 10(-8) M increased the negative ellipticity at 220 nm, suggesting an increase in the alpha-helical content of actin. These results suggest that goniodomin A increases and decreases actomyosin ATPase activity, probably through the stimulatory and inhibitory sites on actin, respectively. It is also suggested that the troponin/tropomyosin complex binds to actin to inhibit the goniodomin A-induced enhancement of actomyosin ATPase activity, probably by affecting the stimulatory site on the molecule.

Tropomodulin isolated from rabbit skeletal muscle inhibits filament formation of actin in the presence of tropomyosin and troponin.

Tropomodulin is a tropomyosin-binding protein, originally isolated from human erythrocytes. Tropomodulin is currently regarded as the sole actin pointed-end capping protein [Weber, A., Pennise, C.R., Babcock, G.G. & Fowler, V.M. (1994) J. Cell Biol. 127, 1627-1635]. This work first describes a procedure for the purification of tropomodulin from rabbit skeletal muscle. Tropomodulin almost completely inhibited filament formation of actin in the presence of tropomyosin and troponin. For the maximal inhibition of actin polymerization, approximately 0.10, 0.12 and 0.003 mol of tropomyosin, troponin and tropomodulin per mol of actin were required, respectively. Fluorescence-intensity measurements, electron-microscopy and sedimentation experiments revealed that only very short fragments and amorphous aggregates, but not filaments, were formed when actin was copolymerized with tropomyosin, troponin and tropomodulin by the addition of 50 mM KCl at pH 8.0. The effects of tropomyosin, troponin and tropomodulin were more remarkable on Ca-actin than on Mg-actin. It appears that tropomodulin caps both the pointed and barbed ends of tropomyosin- and troponin-bound actin filaments.

Functional analysis of human cardiac troponin by the in vitro motility assay: comparison of adult, foetal and failing hearts.

OBJECTIVE: Human cardiac development and heart failure are associated with altered troponin isoform expression and phosphorylation. As the functional effects of these changes in troponin are unknown, we isolated troponin from human foetal, normal adult and failing adult hearts and investigated their regulatory function. METHODS: Human cardiac troponin was assayed for regulatory function by in vitro motility assay and for protein content by SDS PAGE and immunoblotting. RESULTS: Human cardiac troponin regulated movement of actin-tropomyosin filaments over a bed of immobilised heavy meromyosin. At pCa 9, troponin from foetal and adult hearts reduced the fraction of filaments moving from 90% to less than 15% with a modest (25-30%) decrease in velocity. At pCa 5, troponin from normal adult hearts increased filament velocity by up to 47 +/- 3% with no change in the fraction of filaments moving. Foetal troponin increased velocity by only 4 +/- 6% and the effect of troponin from failing hearts was between these values at 31 +/- 5%. Foetal hearts showed different troponin I and T isoform expression compared with adult hearts. No differences in troponin isoform expression were demonstrated between normal and failing adult hearts. CONCLUSIONS: Functioning troponin and tropomyosin may be isolated from human heart and their properties investigated by in vitro motility assay. Both functional and isoform expression differences exist between foetal and adult cardiac troponin. The regulatory function of troponin from adults with end stage heart failure is different from normal adult troponin. These data suggest a role for altered troponin function in human cardiac development and heart failure.