Synthetic aporphine alkaloids are potential therapeutics for Leigh syndrome

Abstract

Mitochondrial diseases are mainly caused by dysfunction of mitochondrial respiratory chain complexes and have a variety of genetic variants or phenotypes. There are only a few approved treatments, and fundamental therapies are yet to be developed. Leigh syndrome (LS) is the most severe type of progressive encephalopathy. We previously reported that apomorphine, an anti- "off" agent for Parkinson's disease, has cell-protective activity in patient-derived skin fibroblasts in addition to strong dopamine agonist effect. We obtained 26 apomorphine analogs, synthesized 20 apomorphine derivatives, and determined their anti-cell death effect, dopamine agonist activity, and effects on the mitochondrial function. We found three novel apomorphine derivatives with an active hydroxy group at position 11 of the aporphine framework, with a high anti-cell death effect without emetic dopamine agonist activity. These synthetic aporphine alkaloids are potent therapeutics for mitochondrial diseases without emetic side effects and have the potential to overcome the low bioavailability of apomorphine. Moreover, they have high anti-ferroptotic activity and therefore have potential as a therapeutic agent for diseases related to ferroptosis.

Figure 1

Structural formula of 26 apomorphine analogs. We obtained 26 apomorphine analogs from 2,000,000 compounds through screening by structural similarity calculation. D1 is nuciferin, and D15 is apomorphine. D6 is vacant.

Figure 2

Aporphine framework and identification of active hydroxy groups. (A) Aporphine framework. (B) Apomorphine has two hydroxy groups at positions 10 and 11. We synthesized three derivatives: D29, D30 and D31. D30 retains a hydroxy group at position 10 and D31 at position 11. (C) Among the three derivatives, D31 was the only compound with the same anti-BSO-induced cell death activity as apomorphine.

Figure 3

Structural formulae of 20 apomorphine derivatives. The synthesized apomorphine derivatives retained the hydroxy group at position 11 of the aporphine framework, except for D29 and D30.

Figure 4

Dopamine D2 receptor activities of apomorphine analogs/derivatives. Using CHO cells that stable express dopamine D2 receptors, dopamine agonist (DA) activities were evaluated and EC50 values were calculated. NA: no activity (A) Apo, EC50 = 35.1 nM; (B) D1, NA; (C) D2, EC50 = NA; (D) D3, EC50 = 10.4 nM; (E) D4, EC50 = 582 nM; (F) D5, EC50 = 206 nM; (G) D7, EC50 = 809 nM; (H) D8, NA; (I) D9, NA; (J) D10, NA; (K) D11, NA; (L) D12, NA; (M) D13, NA; (N) D14, NA; (O) D15, EC50 = 935 nM; (P) D16, NA; (Q) D17, EC50 = 1040 nM; (R) D18, NA; (S) D19, NA; (T) D20, NA; (U) D21, NA; (V) D22, NA; (W) D23, NA; (X) D24, EC50 = 1955 nM; (Y) D25, NA; (Z) D26, NA; (AA) D27, EC50 = 254 nM; (BB) D28, NA; (CC) D29, NA; (DD) D30, NA; (EE) D31, NA; (FF) D36, NA; (GG) D37, NA; (HH) D38, EC50 = 3651 nM; (II) D39, NA; (JJ) D40, NA; (KK) D41, NA; (LL) D42, NA; (MM) D43, NA; (NN) D45, EC50 = 2634 nM; (OO) D47, NA; (PP) D48, NA; (QQ) D50, NA; (RR) D54, NA; (SS) D55, NA.

Figure 5

Anti-BSO-induced cell death effect of apomorphine analogs/derivatives. For compounds that showed > 50% cell viability in the BSO-coadded assay, the EC50 of each compound was determined by BSO addition at a concentration of 10%-20% cell viability. (A) D3, EC50 = 186 nM; (B) D4, EC50 = 1990 nM; (C) D5, EC50 = 2110 nM; (D) D8, EC50 = 180 nM; (E) D9, EC50 = 393 nM; (F) D10, EC50 = 750 nM; (G) D11, EC50 = 6920 nM; (H) D15, EC50 = 613 nM; (I) D18, EC50 = 492 nM; (J) D19, EC50 = 5180 nM; (K) D20, EC50 = 798 nM; (L) D24, EC50 = 1026 nM; (M) D25, EC50 = 2049 nM; (N) D26, EC50 = 289 nM; (O) D27, EC50 = 221 nM; (P) D31, EC50 = 24 nM; (Q) D36, EC50 = 123 nM; (R) D37, EC50 = 186 nM; (S) D38, EC50 = 165 nM; (T) D39, EC50 = 259 nM; (U) D40, EC50 = 55 nM; (V) D41, EC50 = 648 nM; (W) D42, EC50 = 167 nM; (X) D45, EC50 = 96 nM; (Y) D47, EC50 = 124 nM; (Z) D48, EC50 = 128 nM; (AA) D50, EC50 = 207 nM; (BB) D54, EC50 = 497 nM; (CC) D55, EC50 = 4 nM.

Figure 6

Anti-RSL3-induced cell death effect of apomorphine analogs/derivatives. For four compounds that showed EC50 of < 100 nM the BSO-coadded assay, the EC50 of each compound was determined by RSL3 addition at a concentration of 10%-20% cell viability. D31, D55, D40 have methoxy, ethoxy, and propoxy groups as side chains at position 10 of the aporphine framework, respectively. D31 with a methoxy group and D55 with an ethoxy group showed particularly high anti-RSL3-induced cell death activity. (A) D31, EC50 = 27 nM; (B) D40, EC50 = 231 nM; (C) D45, EC50 = 745 nM; (D) D55, EC50 = 21 nM.

Figure 7

ATP production capacity of LS patient-derived skin fibroblasts. No significant enhancement of ATP production upon administration of apomorphine, D31, D55, or D40.

Figure 8

GDF-15 suppression in RSL3 loaded LS fibroblasts of D31, D55, and D40. Compared to the RSL3-only, the groups co-administered with apomorphine, ferrostatin-1, D31, D55, and D40 showed a significant decrease in the concentration of GDF-15 in cell supernatant of LS patient-derived skin fibroblasts.