Highly Selective SERCA2a Activators: Preclinical Development of a Congeneric Group of First-in-Class Drug Leads against Heart Failure

Abstract

The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na⁺/K⁺ ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na⁺/K⁺ ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na⁺/K⁺ ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.

Figure 1

Chemical structures of (A) istaroxime and its metabolite PST3093, and (B) PST3093 synthetic variants 1–12.

Scheme 1. Synthesis of Compounds 1–12 from the Common Precursors 13 and 14

Reagents and conditions: (a) NaH, (3-carboxypropyl)triphenylphosphonium or (3-carboxypentyl)triphenylphosphonium bromide, DMSO. (b) EDC, EtOH. E/Z 1:2. (c) aq LiOH 1 M, THF. (d) LiHMDS, (3-carboxypropyl)triphenylphosphonium bromide, THF. (e) H₂/Pd–C, EtOAc. (f) triethylphosphonoacetate, NaH, dimethylformamide.

Figure 2

Na⁺/K⁺ ATPase activity inhibition in a dog purified enzyme preparation. All compounds were tested in a concentration range from 10^–9 to 10^–4 M in comparison to ouabain. (•) Cpd 9 (−62% at 10^–4 M), (⧫) Cpd 3 (−55% at 10^–4 M), and all the other compounds showed <40% inhibition at 10^–4 M (N = 2).

Figure 3

Screening of the compounds by testing their activity on SERCA2a Ca²⁺ dependency in guinea pig microsomal preparations. Concentration dependency of SERCA2a Ca²⁺ affinity (K_dCa) modulation by all compounds and PST3093 (N = 4–11). All compounds were tested at the concentrations of 10 and 100 nM. PST3093, compounds 5 and 8 were also tested at 1 nM (see text). Data are the mean ± SEM. *p < 0.05 (one-way RM ANOVA plus post hoc Tukey’s multiple comparisons or paired t-test).

Figure 4

Effects of compounds 5 and 8 on SERCA1 ATPase activity and its PLN dependency in guinea pig microsomal preparations. Concentration dependency of SERCA1–Ca²⁺ affinity (K_dCa) modulation with compounds 5 and 8 in skeletal muscle microsomes containing SERCA1 alone (A) and after reconstitution with PLN_1–32 fragments (B) (N = 5). Data are the mean ± SEM. *p < 0.05 (one-way RM ANOVA plus post hoc Tukey’s multiple comparisons or paired t-test).

Figure 5

Modulation of SERCA2a ATPase activity in diseased cardiac preparations. Effect of compounds 5 (A) and 8 (B) (concentration range from 100 to 1000 nM) on SERCA2a maximal activity (V_max) in cardiac homogenates from diabetic (STZ) rats (N = 4–10). Data are the mean ± SEM. *p < 0.05 (one-way RM ANOVA or paired t-test).

Figure 6

Modulation of SR Ca²⁺ uptake under NCX inhibition in V-clamped myocytes from STZ hearts. (A) Na⁺/K⁺ ATPase current (I_NaK) inhibition by compound 5 (n = 22) in rat LV myocytes; I_NaK recording at increasing concentrations of compound 5 and finally to ouabain (OUA as reference) is shown on the left. Data are the mean ± SEM. (B) Effect of compound 5 on SR Ca²⁺ loading in patch-clamped STZ myocytes. SR Ca²⁺ loading by a train of V-clamp pulses was initiated after caffeine-induced SR depletion; NCX was blocked by Na⁺ substitution to identify SERCA2a-specific effects (see Methods and Figure S2); N = 3, ctrl n = 14, with compound 5, n = 11. Panels from left to right: Ca_T amplitude, ER gain (the ratio between Ca_T amplitude and Ca²⁺ influx through I_CaL), and the time constant (τ) of Ca_T decay. *p ≤ 0.05 for the “interaction factor” in RM two-way ANOVA, indicating a different steepness of curves.

Figure 7

Effect of compound 5 on in vivo echocardiographic parameters in STZ diabetic rats. Compound 5 was i.v. infused (0.2 mg/kg/min) in rats 8 weeks after STZ treatment. Echocardiographic parameters were measured before (basal) and at 15 and 30 min during drug infusion and 10 min after drug interruption under urethane anesthesia. Data are the mean ± SEM; all the results of echocardiographic indexes are summarized in Table S2; N = 13; *p < 0.05 (one-way RM ANOVA).