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Background: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. Methods: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). Results: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. Conclusions: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents.

Lo scompenso cardiaco è una delle principali cause di morte improvvisa nei paesi sviluppati ed è noto che il cuore scompensato sia caratterizzato da una ridotta contrattilità causata da un’alterazione della dinamica del calcio tra il sarcoplasma e il reticolo sarcoplasmatico. In questo ambito, istaroxime è un farmaco in fase 2 preclinica che combina l’inibizione della pompa Na+/K+ e la stimolazione di SERCA2a, mostrando effetti promettenti per il trattamento dello scompenso cardiaco acuto. Tuttavia, l’uso di istaroxime è limitato al trattamento i.v. a causa della sua breve emivita (⁓ 1 h) e dalla sua estensiva metabolizzazione a livello epatico portando alla formazione di una molecola chiamata PST3093. Il primo obiettivo del mio progetto di tesi mirava allo studio del PST3093, il principale metabolita di istaroxime, per capire se fosse dotato di attività farmacologica e per comprendere meglio gli effetti in vivo dell’istaroxime. Sulla base dei risultati ottenuti, il secondo obiettivo era quello di sviluppare degli analoghi del PST3093, stimolatori di SERCA2a e dotati di un gruppo metabolicamente stabile per la somministrazione orale. Effetti in vivo e in vitro dei derivati del PST3093 venivano valutati in un modello di ratto con cardiomiopatia diabetica indotta da streptozotocina (STZ), in quanto caratterizzato da disfunzione diastolica associata a down-regolazione di SERCA2a. Innanzitutto, venivano studiati gli effetti del PST3093 sull’attività di SERCA2a e della pompa Na+/K+, sulla dinamica del calcio intracellulare in miociti ventricolari isolati e sull’emodinamica nei ratti STZ. A differenza di istaroxime, il PST3093 risultava uno stimolatore “selettivo” di SERCA2a, privo di effetti sulla pompa Na+/K+ ̧ con un profilo meno aritmogenico del composto di origine. Il PST3093 risultava attivo a concentrazioni nanomolari in preparazioni cardiache di cavia e di ratto STZ e, similmente a istaroxime, stimolava SERCA2a solo in presenza del fosfolambano, diminuendon, Heart failure (HF) is one of the leading causes of sudden death in developed countries and it is known that failing hearts are characterized by reduced contractile properties caused by impaired Ca2+ cycling between the sarcoplasm and sarcoplasmic reticulum (SR). In this field, istaroxime is a small-molecule drug under phase 2 clinical trial that, combining inhibition of Na+/K+ ATPase and SERCA2a stimulation, shows an interesting profile for acute HF treatment. However, istaroxime use is restricted to acute i.v. infusion because of its plasma half-life of about 1 hour in humans and its extensive hepatic metabolism to a molecule, named PST3093. The first aim of my thesis project dealt with the investigation whether PST3093, the main metabolite of istaroxime, may, on its own, be endowed with pharmacological activity and at least partially explain in vivo istaroxime effects. In light of the results, the second aim was to develop PST3093 analogues with metabolically stable groups, with the purpose to generate orally administrable SERCA2a stimulators. In vivo and in vitro effects of PST3093 follow-on compounds were evaluated by using streptozotocin (STZ)-treated rats developing diabetic cardiomyopathy with diastolic dysfunction associated to SERCA2a downregulation. Firstly, we characterized PST3093 effects on SERCA2a and Na+/K+ ATPase activities, intracellular Ca2+ dynamics in isolated ventricular myocytes and in vivo hemodynamic effects in STZ rats. At variance with its parent compound, PST3093 is a “selective” (i.e. devoid of Na+/K+ ATPase inhibition) SERCA2a stimulator, showing a safer profile than istaroxime. It is active at nanomolar concentrations in cardiac preparations from normal guinea pig and STZ rats and, similarly to istaroxime, it stimulates SERCA2a only in the presence of phospholamban (PLN), thus relieving its inhibitory activity on SERCA2a. In-vivo PST3093 i.v. infusion (acute effects) in STZ rats improved overall cardiac performance and reversed most STZ

Aims: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. Methods and results: Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. Conclusions: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Translational perspective: Defi

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues’ side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.