Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays

Abstract

The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function.

Emerging Concepts of Mechanisms Controlling Cardiac Tension: Focus on Familial Dilated Cardiomyopathy (DCM) and Sarcomere-Directed Therapies

“…In contrast with the inotrope dobutamine that increased cAMP and was energywasting, TA1 had no effects on the phosphocreatine/ATP ratio, or ∆G ~ATP . Another study developed a high-throughput assay probing effects of chemical libraries on the cTnI-cTnC interaction with fluorescent probes [103]. Results identified NS5806 as a compound that forms a complex with the N-lobe of cTnC promoting Sw peptide binding without affecting Ca 2+ binding.…”

Fluorescence polarization immunoassay for the determination and screening of medicines.

“…Однако клиническое применение препаратов, воздействующих на миофиламенты, ограничено, а дальнейший прогресс сдерживается неполным пониманием функции миофиламентов на молекулярном уровне и технологиями скрининга малых молекул, точно воспроизводящих эту функцию in vitro. Авторам удалось разработать и охарактеризовать новые высокопроизводительные платформы для скрининга низкомолекулярных эффекторов, нацеленных на взаимодействие между субъединицами тропонина С и тропонина I сердечного тропонинового комплекса [17]. Анализы на основе поляризации флуоресценции использовались для скрининга коммерчески доступных библиотек соединений, и был идентифицирован новый сенсибилизатор кальция, который способен стабилизировать активный тропонин.…”

Synthesis and Biophysical Characterization of Fingolimod Derivatives as Cardiac Troponin Antagonists

“…To overcome this, we have developed in vitro screening assays that allow for the identification of both activators and inhibitors of the NcTnC–cTnI SP interface , and have identified fingolimod as an antagonist that decreases myocardial calcium sensitivity by reducing the affinity of NcTnC for cTnI SP (Figure C). However, fingolimod also increases the Ca 2+ affinity of isolated NcTnC, which is a common feature of troponin-directed small-molecule effectors, likely by stabilizing its open conformation. , We proposed that the balance between NcTnC’s affinity for cTnI SP and Ca 2+ determines whether a compound acts as a cardiac muscle activator or inhibitor.…”