The EF-hand Ca 2+ sensor protein S100A1 has been identified as a molecular regulator and enhancer of cardiac performance. The ability of S100A1 to recognize and modulate the activity of targets such as SERCA2a (sarcoplasmic reticulum Ca 2+ ATPase) and RyR2 (ryanodine receptor 2) in cardiomyocytes has mostly been ascribed to its hydrophobic C-terminal α-helix (residues 75–94). We hypothesized that a synthetic peptide consisting of residues 75 through 94 of S100A1 and an N-terminal solubilization tag (S100A1ct) could mimic the performance-enhancing effects of S100A1 and may be suitable as a peptide therapeutic to improve the function of diseased hearts.
We applied an integrative translational research pipeline ranging from in silico computational molecular modeling and in vitro biochemical molecular assays as well as isolated rodent and human cardiomyocyte performance assessments to in vivo safety and efficacy studies in small and large animal cardiac disease models.
We characterize S100A1ct as a cell-penetrating peptide with positive inotropic and antiarrhythmic properties in normal and failing myocardium in vitro and in vivo. This activity translates into improved contractile performance and survival in preclinical heart failure models with reduced ejection fraction after S100A1ct systemic administration. S100A1ct exerts a fast and sustained dose-dependent enhancement of cardiomyocyte Ca 2+ cycling and prevents β-adrenergic receptor–triggered Ca 2+ imbalances by targeting SERCA2a and RyR2 activity. In line with the S100A1ct-mediated enhancement of SERCA2a activity, modeling suggests an interaction of the peptide with the transmembrane segments of the sarcoplasmic Ca 2+ pump. Incorporation of a cardiomyocyte-targeting peptide tag into S100A1ct (cor-S100A1ct) further enhanced its biological and therapeutic potency in vitro and in vivo.
S100A1ct is a promising lead for the development of novel peptide-based therapeutics against heart failure with reduced ejection fraction.
Peptide therapeutics have played a notable role in medical practice since the advent of insulin therapy in the 1920s. There are >100 US Food and Drug Administration– and European Medicines Agency–approved peptide medicines on the market. Peptidic drugs are increasingly appreciated because of their on-target selectivity, specificity, potency, and safety. Polypeptides and their targets have coevolved to such extent that it is difficult to design an artificial molecular entity, such as a small molecule, that substantially mimics the accuracy of fit and coverage of peptidic structures at their target sites. To date, >170 peptides are in active clinical development, with many more in preclinical studies, which may result in broad clinical use of peptide therapeutics across all medical disciplines, including cardiovascular diseases, in the years to come.
It is against this background that we report the development of the S100A1ct peptide as a potential therapeutic agent for systemic treatment against heart failure with reduced ejection fraction (HFrEF). S100A1ct is a short synthetic peptide comprising the C-terminal helix (amino acids [aa] 75–94) of the native human S100A1 protein and a 6-residue N-terminal tag to aid chemical synthesis and solubility. The parent protein S100A1 (1–94 aa) is a homodimer and a member of the EF-hand calcium (Ca 2+) sensor protein superfamily, which comprises at least 20 paralogs. S100A1 is expressed in a tissue- and cell-specific manner with highest abundance in cardiac and skeletal muscle, where it resides at the sarcoplasmic reticulum (SR), the sarcomere, and within the mitochondria of myocytes. Numerous in vitro and in vivo genetic gain- and loss-of-function studies by us and others have shown that S100A1 plays a decisive role as a molecular enhancer of heart and skeletal muscle contractile performance. This is attributable in part to the ability of S100A1 to bind to and regulate the activity of a number of key molecular effectors, including SERCA2a (SR Ca 2+ ATPase), RyR (ryanodine receptor) 1 and 2, F1-ATPase (mitochondrial ATP synthase), and titin, all of which govern cardiac and skeletal muscle Ca 2+ cycling, energy supply, and mechanical properties.
Nuclear magnetic resonance and crystallographic determination of the 3-dimensional structure and dynamics of S100A1 indicated that a hydrophobic binding pocket involving its C-terminal helix becomes exposed upon Ca 2+ binding, providing a possible explanation for the Ca 2+-dependent interactions of S100A1 with its intracellular targets. Computational modeling identified the hydrophobic residues in the α-helical C-terminus of S100A1 as able to interact, for example, with their respective molecular epitopes on the RyR1. Comparative molecular assays using chemically permeabilized skeletal muscle fibers consistently demonstrated an equipotent enhancement of RyR1-dependent SR Ca 2+ release and isometric force development by human recombinant S100A1 protein and by the S100A1ct peptide. Further experiments, using chemically permeabilized ventricular cardiomyocytes, showed a similar rate of attenuation of diastolic RyR2 Ca 2+ spark frequency by the S100A1 protein and the S100A1ct peptide, as well as the ability of the peptide to not only mimic the activated “on” state of the parent protein but to outcompete the binding of S100A1 at the RyR2 receptor.
These in vitro results provide the starting point for the systematic in vitro and in vivo investigation of the biological and therapeutic properties of S100A1ct, which resulted in the clinically relevant discovery that the synthetic peptide, owing to its cell-penetrating ability, is a promising lead for the development of peptide-based therapeutics against HFrEF. These results were achieved by using a vertical translational research approach spanning from systematic experimental investigation and computational modelling of S100A1ct actions and interactions in molecular model systems to in vitro and in vivo S100A1ct treatment studies in failing human and rodent cardiomyocytes from HFrEF hearts and in mouse and pig HFrEF models.
Thus, our translational study provides the foundation to develop an S100A1ct peptide-based drug toward safe and efficacious HFrEF treatment in a first-in-human study with a convenient dosing regimen.
Additional resources and data supporting the findings in this article are available from the corresponding author upon reasonable request. A more detailed description of each method and protocol is provided in the Supplemental Material. All animal procedures and experiments were carried out according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and were approved by the regional or local institutional animal care and use committee of Baden-Württemberg, Germany, and Jefferson University, Philadelphia, PA, respectively.
All mice, rats, and rabbits were housed in institutional standard cages at 22 °C with a 12-hour light/dark cycle with free access to water as well as standard chow (mice and rats) or commercial diet (high-fiber rabbit diet). Bay cage types were used for housing pigs. Per pig, a body weight (BW)–prescribed floor space of at least 0.5×0.7 m 2 was provided according to directive 2010/63/EU with straw bedding. The environment was enriched by pellet balls, chains, and gnawing rods. Humidity and temperature were kept at 50% to 60% and 20 °C to 24 °C, respectively. Access to water was unlimited and restricted food was provided twice a day (SAF130M). ARRIVE (Animal Research: Reporting of In Vivo Experiments) reporting guidelines were used to describe in vivo studies involving laboratory animals. For further detail, please refer to the Supplemental Methods (ARRIVE criteria for in vivo studies 1 through 5).
Recombinant human S100A1 (rhS100A1) protein was generated and purified as described previously. Synthetic S100 and control peptides were custom-made by commercial suppliers (Eurogentec, GeneScript, and Bachem); sequences are given in Figure 1A, Figure S1A and S1C, and Figure 5A. In general, S100A1-derived peptides were first tested against control (scrambled peptide) and vehicle (solvent only). Because the control peptide showed no difference from vehicle in any experiment of our study, we compared the effect of S100A1-derived peptides on vehicle only. After establishment of the in vitro dose–response relationship on calcium cycling, subsequent experiments were limited to a 1-μM peptide concentration.
