Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure

Abstract

Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure.

Figure 1. N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes.

In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. (a) Chemical structure of SERCA2a SUMOylation activator. N106, N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. (b-g) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant (τ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n=25 cardiomyocytes from each three of hearts per condition. (h) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. (i) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. (j) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. (n=3 mice hearts per each condition). Data are represented as mean±s.e.m. *P≤0.05; **P<0.001 (Student's t-test) from DMSO control.

Figure 2. Time-course experiments with N106 in isolated adult cardiomyocytes.

(a–f) Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with 10 μM of N106 compound over a time course as indicated. The time course of the contractile response to N106 was determined as peak shortening (a), the time to maximal velocity of sarcomere shortening (b) and re-lengthening (c). The time-course changes in calcium kinetic parameters such as calcium transient (d), decay time constant (e) and the time to 90% baseline fluorescence in cardiomyocytes (f) were detected. n=25 cardiomyocytes from each three of hearts per condition. DMSO was used as vehicle control. The time courses of the ATPase activity of SERCA2a (g), level of SUMOylated SERCA2a (h) and level of SUMO-1 conjugates (i) were determined by using the same set of cardiomyocytes (two independent experiments). Data are represented as mean±s.e.m. *P≤0.05; **P<0.001 (Student's t-test) from DMSO control.

Figure 3. SUMO-specific activating enzyme, E1, is a direct target of N106.

Mechanism of action of N106 on individual steps in the SUMOylation cascade in vitro. (a) Inorganic pyrophosphate resulting from ATP hydrolysis in E1-catalysed SUMO-1 activation in the presence of increasing concentration of N106 compound was assessed by fluorogenic pyrophosphate assay kit. Recombinant human SUMO-specific E1 (0.05 μM), human Ubc9 (0.25 μM), human SUMO-1 (12.5 μM) and ATP (1 mM) were incubated with increasing concentration of N106 for 30 min at 37 °C and quantified using a fluorescence microplate reader. (b) The SAE2-SUMO-1 conjugation was detected followed by western blotting with anti-SAE2 antibodies. SUMO E1 (0.05 μM), SUMO-1 (12.5 μM) and ATP (1 mM) were co-incubated with increasing concentration of N106 for 30 min at 37 °C. The reaction mixtures were separated on 7.5% SDS–PAGE gel under non-reducing conditions. (c) E2-SUMO-1 conjugation was visualized by western blotting with anti-Ubc9 antibody. SUMO E1 (0.05 μM), Ubc9 (0.25 μM), SUMO-1 (12.5 μM) and ATP (1 mM) were incubated without or with increasing concentration of N106 for 30 min at 37 °C. SUMO-specific E1-dependent E2-SUMO-1 conjugation was visualized by western blotting with anti-Ubc9 antibodies. (d) Ribbon representation for the SUMO E1/SUMO-1-N106. Atoms for the N106, SUMO-1 (yellow) and SUMO E1 domains are shown (SAE1 in grey and SAE2 consists of three domains such as ubiquitin-folding domain (UFD) in green, adenylation domain in blue and cysteine domain in red). The structure of the human SUMO-activating enzyme, PDB Code 3kyd was used as a model. Residues target mutagenesis in putative binding pocket for N106 to SUMO-activating enzyme are shown in close-up images (glutamine 312 in green and valine 315 in purple). Alternations in the putative binding pocket decrease the N106-mediated biochemical activity of SUMO E1. (e) ATP hydrolytic activity of WT and mutant SUMO E1 are determined. (f) The E1-SUMO-1 conjugation was detected followed by western blot analysis with anti-SAE2 antibody. *The SAE2-SUMO-1 conjugated bands. (g) The E1-dependent Ubc9-SUMO-1 conjugation was visualized by western blotting with anti-Ubc9 antibody. *The E2-SUMO1-conjugating bands. Three independent experiments were performed. Data are represented as mean±s.e.m. WB, western blotting.

Figure 4. Infusion of N106 induces a sustained reversal of contractility in HF mouse.

Beneficial effects of acute N106 treatment on cardiac function were shown in pressure overload-induced mouse model of HF. (a) Representative pressure volume loop showing that haemodynamic function of each increasing doses (0.1, 1.0 and 10 mg kg⁻¹) of N106 compared with the baseline (with vehicle) at 30 min post treatment. Baseline in black; 0.1 mg kg⁻¹ N106 compound in green; 1 mg kg⁻¹ N106 compound in blue; 10 mg kg⁻¹ N106 compound in red. (b–d) The ESPVR slope was steeper and maximal rate of contractility (dP/dt_max) was increased when the heart received the N106 compound on dose-dependent manner. The relaxation parameter (τ) also enhanced in these conditions (n=10 independent mice per each N106 compound dose condition). (e) The effect of 10 mg kg⁻¹ of N106 infusion on SERCA2a protein level was determined by western blot analysis. GAPDH (glyceraldehyde 3-phosphate) was used as a loading control. (f) SUMOylated SERCA2a level was examined by immunoprecipitation followed by western blot analysis. Data are represented as mean±s.e.m. *P≤0.05; ** P<0.001 (Student's t-test) versus baseline. WB, western blot analysis; NS, not significant.

Figure 5. Lack of effects of N106 on haemodynamic parameters in cardiac-specific Serca2a knockout mice.

Systolic and diastolic function was determined in conditional cardiac-specific Serca2a knockout mice by pressure–volume analysis. Four weeks after tamoxifen administration, the mice showed severe HF phenotype. There was no significant change in haemodynamic parameters with N106 treatment after a single dose (1 mg kg⁻¹) (a-f) and different doses (0.1, 1 and 10 mg kg⁻¹) (g,h). Data are represented as mean±s.e.m. n=3∼8 mice per each condition. NS, not significant; Student's t-test.