New Insights into the Structure-Activity Relationship and Neuroprotective Profile of Benzodiazepinone Derivatives of Neurounina-1 as Modulators of the Na+/Ca2+ Exchanger Isoforms

Abstract

Due to the neuroprotective role of the Na⁺/Ca²⁺ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1-19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.

Figure 1

Chemical structure of Neurounina-1.

Scheme 1. Reagents and Conditions: (a) Formaldehyde Solution 37% wt in H₂O (10 equiv), Pyrrolidine (Compounds 1 and 2; 10 equiv) or Piperidine (Compound 3; 10 equiv), and Glacial Acetic Acid, MW (500 W, 15 min; T = 80 °C)

Scheme 2. Reagents and Conditions: (a) NaH (60% Dispersion in Mineral Oil), 0 °C, DMF then 4 h, rt; (b) ACy (i–viii), K₂CO₃ (1.1 equiv), NaI (1.1 equiv), CH₃CN, Reflux, 3 h

Scheme 3. Reagents and Conditions: (a) NaH (60% Dispersion in Mineral Oil), 0 °C, DMF and Then 4 h, rt; (b) Anhydrous CH₂Cl₂/TFA (8:2, v/v, 20 mL), rt, 1 h; (c) ACy (i–iv and vii–viii), TBTU (1.1 equiv), HOBt (1.1 equiv), DIPEA, DMF, Overnight, rt; and (d) (1) SOCl₂ (10 equiv), Anhydrous CH₂Cl₂, Reflux, 1 h and (2) ACy (v and vi, 2 equiv), Anhydrous Tetrahydrofuran, Overnight, rt

Figure 2

High-throughput screening of the novel benzodiazepinone derivative efficacy on NCX activity in the reverse mode in BHK-NCX1 and BHK-NCX3 cells loaded by Fluo-4. (A) Effect of compound 14 (10 nM and 10 μM), compound 1 (10 nM and 10 μM), and Neurounina-1 (10 nM) on NCX1 in BHK-NCX1 cells. *p < 0.05 vs control. (B) Effect of the same compounds on NCX3 in BHK-NCX3 cells. *p < 0.05 vs control, Neurounina-1, and compound 1. (C) Effect of two different concentrations of compound 4 on the NCX1 reverse mode in BHK-NCX1 cells. *p < 0.05 vs Neurounina-1 and internal control (untreated cells). The effects of compounds 1–19 on NCX1 and NCX3 reverse mode activity measured in BHK-NCX1 and BHK-NCX3 loaded by Fura-2 AM have been reported in Figure S1.

Figure 3

Effect of compound 14 on NCX1 activity in BHK-NCX1 cells. (A,B) Representative superimposed traces and quantification of the effect of compound 14 on Na⁺-free-induced [Ca²⁺]_i increase through NCX1 reverse mode of operation in Fura-2-loaded BHK-NCX1 cells. The bar graph reports the mean ± SEM of the maximal [Ca²⁺]_i responses measured in approximately 60 cells per group. Averaged data from four different experimental sessions were normalized as the percentage of controls. *p < 0.05 vs control and 1 nM; **p < 0.05 vs all. (C,D) Representative I_NCX traces recorded by whole-cell patch-clamp electrophysiology in control cells and in cells treated with compound 14 (10 nM). The bar graphs in (D) report the mean ± SEM of the forward and reverse NCX1 current densities, respectively, measured in at least 10 cells for each experimental group. Reverse I_NCX amplitude was measured at +60 mV, while forward I_NCX was measured at −120 mV. *p < 0.05 vs each internal control.

Figure 4

Effect of compound 14 on NCX3 activity in BHK-NCX3 cells. (A,B) Representative superimposed traces and quantification of the effect of compound 14 on Na⁺-free-induced [Ca²⁺]_i increase through NCX3 reverse mode of operation in Fura-2-loaded BHK-NCX3 cells. The bar graph reports the mean ± SEM of the maximal [Ca²⁺]_i responses measured in approximately 50 cells per group. Averaged data from four different experimental sessions were normalized as the percentage of controls. *p < 0.05 vs control and 1 μM; **p < 0.05 vs all. (C,D) Representative I_NCX traces recorded by whole-cell patch-clamp electrophysiology in control cells and in cells treated with compound 14 (10 μM). The bar graphs in (D) report the mean ± SEM of the forward and reverse NCX3 current densities, respectively, measured in nine cells for each experimental group. Reverse I_NCX amplitude was measured at +60 mV, while forward I_NCX was measured at −120 mV. *p < 0.05 vs each internal control.

Figure 5

Effect of compound 4 on NCX1 and NCX3 activity in BHK-NCX1 and BHK-NCX3 cells. (A) Representative I_NCX traces recorded in BHK-NCX1 by whole-cell patch-clamp electrophysiology in control cells and in cells treated with compound 4 (10 μM). The bar graphs in (A) report the mean ± SEM of the reverse and forward NCX1 current densities, respectively, measured in 10 cells for each experimental group. Reverse I_NCX amplitude was measured at +60 mV, while forward I_NCX was measured at −120 mV. *p < 0.05 vs each internal control. (B) Representative I_NCX traces recorded in BHK-NCX3 by whole-cell patch-clamp electrophysiology in control cells and in cells treated with compound 4 (10 μM). The bar graphs in (B) report the mean ± SEM of the reverse and forward NCX1 current densities, respectively, measured in 10 cells for each experimental group. Reverse I_NCX amplitude was measured at +60 mV, while forward I_NCX was measured at −120 mV. (C) Quantification of the concentration-dependent effect of compound 4 on Na⁺-free-induced [Ca²⁺]_i increase through NCX1 reverse mode of operation in Fura-2-loaded BHK-NCX1 cells. The bar graph reports the mean ± SEM of the maximal [Ca²⁺]_i responses measured in approximately 30 cells per group. Averaged data from four different experimental sessions were normalized as the percentage of controls. *p < 0.05 vs control, 1, and 1 0 nM; **p < 0.05 vs all.

Figure 6

Neuroprotective effect of compound 1 in primary cortical neurons exposed to OGD followed by RX or chemical hypoxia followed by RX. (A) Bar graph depicting the effect on cell viability of compounds 1 (10 nM), 14 (10 μM), and 4 (10 μM) added during both OGD and RX. Cell viability was measured at the end of RX. Data are means ± SEM of five separate experiments and are reported as the percentage of cell viability in the control (100% viability). *p < 0.05 vs normoxic cells (control); **p < 0.05 vs OGD/Rx. (B) Bar graph depicting the effect on cell viability of compound 1 preincubated before OGD + RX. After preincubation, compound 1 was removed during OGD + RX and it was compared to ischemic PC. *p < 0.05 vs normoxic cells (control); **p < 0.05 vs OGD/Rx. (C) Bar graph depicting the effect on cell viability of compound 1 (10 nM) added during both chemical hypoxia (Ch Hyp, 45′) and RX (3 h). Cell viability was measured at the end of RX. Data are means ± SEM of three separate experiments and are reported as the percentage of cell viability in the control (100% viability). *p < 0.05 vs normoxic cells (control); **p < 0.05 vs Ch Hyp + Rx.

Figure 7

Lowest energy conformers of Neurounina-1 and compounds 1–19 superimposed by the heavy atoms of the benzodiazepinone ring system. (A) Compounds with aliphatic R groups: Neurounina-1, compounds 1–3, 4–7, and 12–15. (B) Compounds with an aromatic R group: 8–11 and 16–19. Carbon atoms are colored according to the spacer group: Neurounina-1, compounds 1–3 (carbon atoms: green), 4–11 (carbon atoms: orange), and 12–19 (carbon atoms: magenta). The active analogues (Neurounina-1, compounds 1, 4, and 14) are displayed as ball and sticks; the inactive analogues are displayed as sticks. Heteroatoms are colored by atom type; hydrogen atoms are omitted for clarity of presentation except for those involved in hydrogen bonds. Hydrogen bonds are displayed as green dashed lines.

Figure 8

(A) Neurounina-1 and compound 1 (NCX1 activators); (B) compound 14 (NCX1/3 activator); and (C) compound 4 NCX1 (inhibitor); pharmacophore features: aromatic ring (centroid and plane; green), hydrophobic-aliphatic group (centroid; blue), hydrogen bond acceptor head/tail (balls and arrows; green), distances (Å; black). (D–F): shape features. All structures are superimposed by the carbon atoms of the benzodiazepinone moiety and displayed as ball and sticks. Heteroatoms are colored by atom type: carbon atoms are colored: Neurounina-1 and compound 1, green; compound 4, orange; and compound 14, magenta. Hydrogen atoms are omitted for the clarity of presentation.

Figure 9

(A) Calculated Neurounina-1 lowest energy conformation (carbons: pink) superimposed on the X-ray structure of I-BET (carbons: dark gray) in complex with the BRD4 bromodomain (PDB ID: 3P5O). The I-BET binding site is colored in red (BRD4_BDZ_BS_1) and cyan (BRD4_BDZ_BS_2). P85 is evidenced in CPK. The pyrrolidine substituent of Neurounina-1 is indicated by a black arrow. (B) Sequence alignments of the α1 and α2 repeat regions suggested to be involved in Neurounina-1 binding with the BRD4 alprazolam binding site. BRD4 residues establishing interactions with the benzodiazepine ligand are evidenced with red squares. (C) Calculated Neurounina-1 lowest energy conformation (carbons: pink) superimposed on the X-ray structure of flurazepam (carbons: green) in complex with ELIC (PDB ID: 2YOE). The binding site is colored in red (ELIC_BDZ_BS). P82 is evidenced in CPK. The pyrrolidine substituent of Neurounina-1 is indicated by a black arrow. (D) Sequence alignments of the α1 and α2 repeat regions suggested to be involved in Neurounina-1 binding with the ELIC flurazepam binding site. ELIC residues establishing interactions with the benzodiazepine ligand are evidenced with red squares. NCX1 P848, BRD4 P82, and ELIC P85 proline residues are evidenced and labeled.

Figure 10

(A,B). Molecular interaction model between Neurounina-1 and NCX_Mj resulting from our bioinformatic and structural analysis. The X-ray structure of the NCX_Mj transporter in the sodium-loaded semi-open conformation (PDB ID: 5HWY) is colored in white with NCX1_alpha1_Neu and NCX1_alpha2_Neu evidenced in cyan and yellow, respectively. The protein structure is displayed as follows: helical structures as wide cylinders, β-sheets as arrows, and coil and turn regions as tubes. The sodium atoms are displayed in ball and stick and colored in violet. The putative bioactive conformer of Neurounina-1 is displayed in ball and stick and colored by atoms (C = green, O = red, and N = blue). Neurounina-1 solvent accessible surface is showed and colored in white/transparent. Proline P212 is evidenced in CPK and colored in orange. (C) Sequence alignments of the α1 and α2 repeat regions of human NCX1 suggested to be involved in Neurounina-1 binding with the corresponding segments of NCX_Mj. NCX_Mj P212 and NCX1 P848 proline residues are evidenced and labeled.