Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity

Abstract

Background and purpose: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor.

Experimental approach: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae.

Key results: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC₅₀ values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (I_Na ) and hERG K_V 11.1 currents (I_hERG ) in a concentration-dependent manner; IC₅₀ values were 23.2 and 10.0 μM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 μM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose.

Conclusion and implications: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.

Keywords: NCX; ORM-11372; cardiac safety; positive inotropic effect; sodium-calcium exchanger.

FIGURE 1

The upper part of the figure shows the pharmacophore model used for virtual screening. In silico hits were further filtered based on predicted activity, calculated logarithm of solubility (clogS), calculated logarithm of partition coefficient (clogP), and diversity. The middle part displays the hit structure and substructure features optimized in parallel processes. At the bottom of the figure, two routes used for the synthesis of the compounds are presented, namely, (1) reductive amination and (2) alkylation under basic conditions

FIGURE 2

Illustrative chemical structures (out of 135 synthesized molecules) during the chemical optimization of the scaffold. ORM‐11372 had the most favourable profile overall, out of the synthesized derivatives

FIGURE 3

Effect of ORM‐11372 on the bidirectional I _NCX of human‐induced pluripotent stem cell (iPSc)‐derived cardiomyocytes. (a) The experimental time course included a ramp voltage protocol to be repeated every 20 s. The current traces at the labels (A: control; B and C: in the presence of 3‐ and 30‐nM ORM‐11372; and D: when I _NCX is fully blocked by 10‐mM NiCl₂) are shown in (b) enlarged and superimposed (with the voltage protocol below) images. (c) The concentration–response curve for ORM‐11372 on the outward (3 nM, n = 4; 10 nM, n = 6; 30 nM, n = 4; and 100 nM, n = 3) and inward (3 nM, n = 4; 10 nM, n = 4; 30 nM, n = 3; and 100 nM, n = 2) I _NCX was determined at +60 and −100 mV respectively. The IC₅₀ values are calculated using non‐linear regression. Data shown are individual values; n refers to the number of iPS derived cardiomyocytes

FIGURE 4

The effect of ORM‐11372 on the reverse sodium–calcium exchanger (NCX) current measured in rat ventricular cardiomyocytes is presented in panel (a). The top left panel (a) shows the voltage protocol applied during experiments: The I–V (current–voltage) relationship of the Na⁺/Ca²⁺ exchanger current was measured through the use of ramp pulses at 20‐s intervals. The ramp pulse initially lead to depolarization from the holding potential of −40 to 60 mV, at a rate of 100 mV·s⁻¹, followed by hyperpolarization to −100 mV, and depolarization back to the holding potential. The middle panel illustrates original current records in the absence (control) and presence of 10‐nM ORM‐11372 and after applying 10‐mM NiCl₂. The Ni²⁺‐sensitive current traces clearly show that 10‐nM ORM‐11372 effectively inhibits the reverse NCX current. Example of the I–V (current–voltage) relationship of the Na⁺/Ca²⁺ exchanger current is shown in panel (b). The magnitude of reverse NCX current measured at 20 mV was reduced by ORM‐11372 in a concentration‐dependent manner in rat ventricular cardiomyocytes in panel (c). The concentration–response curve for ORM‐11372 on the outward (n = 5) and inward (n = 3) I _NCX was determined at +60 and −100 mV respectively. The IC₅₀ value for the outward I _NCX current is calculated using non‐linear regression. Data shown are individual values; n refers to the number of rat primary ventricular cardiomyocytes

FIGURE 5

The effects of selected NCX inhibitors were tested on the forward mode in confirmatory hNCX1 inhibition assays in HEK293 cells. In (a), data shown are means ± SEM from n=5 assays. In (b), experimental records show the effects of increasing concentrations of ORM‐11372 (0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1μM) and in (c), of ORM‐10962 (0.03, 0.1, 0.3, 1, 3 and 10μM) to decrease Ca²⁺ efflux (downward deflection) from control conditions (blue trace). Each assay derived from one plate with four replicates per plate

FIGURE 6

Effects of ORM‐11372 on various cardiac ion channel currents of induced pluripotent stem cell (iPSc)‐derived cardiomyocytes (illustrative figures in panels a–c) and contraction force in guinea pig papillary muscle (panels d–g). (a) The L‐type I _Ca was minimally inhibited by 1‐μM ORM‐11372. (b) The concentration‐dependent inhibition of K_V11.1 current by 0.3‐, 3‐, 10‐, and 30‐μM ORM‐11372 and (c) Na_V1.5 current by 1‐, 3‐, 10‐, and 30‐μM ORM‐11372. The applied voltage protocols are shown as insets. The traces marked by asterisk in (a) and (c) are the currents in the presence of 100‐nM nitrendipine and 2‐mM lidocaine respectively. (d) ORM‐11372 increased twitch tension, which indicates increased SR load. (e) ORM‐11372 did not increase resting tension, that is, no increase in diastolic calcium. (f) ORM‐11372 did not affect time to peak demonstrating that calcium release from ryanodine receptors is normal. (g) Also, half relaxation time was unchanged showing that SERCA function remains normal. Data shown are means ±SEM (n = 5) and individual values with red and blue circles for female and male respectively. Two‐way ANOVA followed by Sidak's multiple comparison test. *P < 0.05, signficant effect of ORM‐11372; two‐way ANOVA followed by Sidak's multiple comparison test. Pacing rate 1 Hz. Temperature 37°C

FIGURE 7

Effects of ORM‐11372 on action potentials (APs) in human ventricular trabeculae (n = 2, two replicates). (a) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle (V) control and after exposure to ORM‐11372 (0.1, 1, and 10 μM) and 0.1‐μM dofetilide (Dof) (the positive control). (b) Mean changes in APD₉₀, triangulation, and short‐term variability analysis of AP duration (STV) values were in cadence when trabeculae were incubated with ORM‐11372 and Dof at 1 and 2 Hz. Notably, the effects of ORM‐11372 and Dof on APD₉₀/triangulation activity and STV are plotted on a separate Y‐axis. Data shown are individual values; n refers to the number of human hearts, and replicates refer to the number of trabeculae in the same heart

FIGURE 8

Effects of ORM‐11372 on haemodynamics in isoflurane‐anaesthetized rats, 7 days after the induction of myocardial infarction (MI, n = 6) or in sham rats (n = 6). Effects on the left ventricular contractility (LV + dP/dt _max) are shown in panel (a), along with values for (b) relaxation (LV − dP/dt _max), (c) heart rate, and (d) systolic arterial BP. Data shown are individual values with means ± SEM; n refers to number of rats. *P<0.05, significantly different from base line (BL); two‐way repeated measures ANOVA followed by Dunnett's post hoc test

FIGURE 9

Effects of ORM‐11372 on haemodynamics in S‐ketamine‐anaesthetized rabbits (n = 5). The effects on (a) left ventricular contractility (LV + dP/dt _max), (b) left ventricular relaxation (LV − dP/dt _max), (c) heart rate, and (d) systolic arterial BP are shown. Data shown are individual values with means ± SEM; n refers to the number of rabbits. *P<0.05, significantly different from base line (BL); one‐way repeated measures ANOVA followed by Dunnett's post hoc test