High Throughput Screen Identifies Small Molecule Effectors That Modulate Thin Filament Activation in Cardiac Muscle

Abstract

Current therapeutic interventions for both heart disease and heart failure are largely insufficient and associated with undesired side effects. Biomedical research has emphasized the role of sarcomeric protein function for the normal performance and energy efficiency of the heart, suggesting that directly targeting the contractile myofilaments themselves using small molecule effectors has therapeutic potential and will likely result in greater drug efficacy and selectivity. In this study, we developed a robust and highly reproducible fluorescence polarization-based high throughput screening (HTS) assay that directly targets the calcium-dependent interaction between cardiac troponin C (cTnC) and the switch region of cardiac troponin I (cTnISP), with the aim of identifying small molecule effectors of the cardiac thin filament activation pathway. We screened a commercially available small molecule library and identified several hit compounds with both inhibitory and activating effects. We used a range of biophysical and biochemical methods to characterize hit compounds and identified fingolimod, a sphingosin-1-phosphate receptor modulator, as a new troponin-based small molecule effector. Fingolimod decreased the ATPase activity and calcium sensitivity of demembranated cardiac muscle fibers in a dose-dependent manner, suggesting that the compound acts as a calcium desensitizer. We investigated fingolimod’s mechanism of action using a combination of computational studies, biophysical methods, and synthetic chemistry, showing that fingolimod bound to cTnC repels cTnISP via mainly electrostatic repulsion of its positively charged tail. These results suggest that fingolimod is a potential new lead compound/scaffold for the development of troponin-directed heart failure therapeutics.

Photocatalytic Hydroaminoalkylation of Styrenes with Unprotected Primary Alkylamines

“… 35 It has also been recently identified as a promising lead for troponin-directed heart failure therapeutics. 36 Several concise synthetic routes to Fingolimod 4 have been developed over the past two decades, 37 but we reasoned that a HAA approach could raise the bar in terms of atom- and step-economy. Gratifyingly, the application of our optimized conditions to serinol 16 and 4-octylstyrene 17 (derived in 1 step from the commercial aldehyde) gave Fingolimod 4 in 43% isolated yield ( Figure 5 A).…”

“…Fingolimod ( 4 ), developed by Novartis, is a S1P 1 receptor agonist used to treat relapsing-remitting multiple sclerosis, with worldwide sales of $3 billion in 2020 . It has also been recently identified as a promising lead for troponin-directed heart failure therapeutics . Several concise synthetic routes to Fingolimod 4 have been developed over the past two decades, but we reasoned that a HAA approach could raise the bar in terms of atom- and step-economy.…”

Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile regulation

“…Our assay adds to a very small number of cTnC fluorescence-based assays conducive to HTS for therapeutic compounds to treat heart failure ( Vetter et al, 2020 ; Parijat et al, 2021 ). At this time, it is unclear which screening platform will be most efficacious.…”

Modular Photocatalytic Synthesis of α-Trialkyl-α-Tertiary Amines

“…Molecules displaying an α-tertiary amine (ATA) motif  a nitrogen with an α-carbon bearing three C–C bonds  show versatile and tunable physicochemical and biological properties, making it an important structural unit that is found in a diversity of pharmaceutical agents and biologically relevant natural products (Figure A). − The fully substituted carbon atom adjacent to the nitrogen atom impacts on amine basicity and lipophilicity, and provides three exit vectors through which interactions with a biological receptor can be tuned. Furthermore, when coupled with the capacity to add up to two further substituents to the nitrogen atom, the presence of an α-tertiary amine can substantially impact the topology of molecules.…”