A small-molecule DS44170716 inhibits Ca2+-induced mitochondrial permeability transition

Abstract

Mitochondria are involved in a variety of physiological and pathological processes. Ca²⁺ uptake is one of the important functions of the organelle for maintenance of cellular Ca²⁺ homeostasis. In pathological conditions such as ischemia reperfusion injury, Ca²⁺ overload into mitochondria induces mitochondrial permeability transition (MPT), a critical step for cell death. Because inhibition of MPT is a promising approach to protecting cells and organs, it is important for drug discovery to identify novel chemicals or mechanisms to inhibit MPT. Here we report upon a small-molecule compound DS44170716 that inhibits Ca²⁺-induced MPT in rat liver isolated mitochondria. DS44170716 protects human liver HepG2 cells from Ca²⁺-induced death with a level of protection similar to cyclosporin A (CsA). The inhibitory mechanism of DS44170716 against MPT is independent on PPIF, a target of CsA. DS44170716 blocks Ca²⁺ flux into the mitochondria by decreasing mitochondrial membrane potential, while potently inhibiting mitochondrial complex III activities and weakly inhibiting complex IV and V activities. Similarly, complex III inhibitor antimycin A, complex IV inhibitor KCN or complex V inhibitor oligomycin inhibits Ca²⁺ uptake of isolated mitochondria. These results show that DS44170716 is a novel class inhibitor of MPT by blocking of mitochondrial complexes and Ca²⁺-overload into mitochondria.

Figure 1

DS44170716 inhibits mitochondrial permeability transition. (a) Structure of DS44170716. (b) Effect of DS44170716 on mitochondrial swelling. Representative temporal profiles are shown. (c) Inhibition rate of mitochondrial swelling by DS44170716. The levels of OD (540 nm) 30 min after application of CaCl₂ were used for the inhibition rate. Inhibition 0% or 100% was defined as CaCl₂-treated vehicle data or no CaCl₂ data, respectively. Data obtained from 3 independent samples are shown as the mean with SEM. (d) Effects of DS44170716 on Ca²⁺-induced death in HepG2 cells. Cell death levels were measured by LDH released from the cells. Cell protection 0% or 100% was defined as the mean of vehicle data or mean of 100 μM CsA, respectively. Raw data are presented in Supplemental Fig. 1. Data obtained from 4 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test).

Figure 2

DS44170716 inhibits mitochondrial swelling in a different mechanism of cyclosporin A. (a) Effect of NIM-811 on swelling in the presence of CsA. (b) Effect of ruthenium red on swelling in the presence of CsA. (c) Effect of DS44170716 on swelling in the presence of CsA. Inhibition 0% or 100% was defined as 500 μM CaCl₂-treated mitochondria (a, Lane 2) or 10 μM CsA-treated mitochondria (a, Lane 3), respectively. Data obtained from 3 independent samples are shown as the mean with SEM. Raw data are presented in Supplemental Fig. 2. (d) No significant binding activity of DS44170716 to cyclosporin A-binding site of PPIF. Binding activities of radio-labeled CsA to PPIF were measured by SPA assay. Data obtained from 3 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test).

Figure 3

DS44170716 inhibits mitochondrial Ca²⁺ uptake in isolated mitochondria from pig heart. (a) Effect of Ru360 on Ca²⁺ uptake activities of isolated mitochondria. (b) Effect of FCCP on Ca²⁺ uptake activities of isolated mitochondria. (c) Effect of DS44170716 on Ca²⁺ uptake activities of isolated mitochondria. (d) Inhibitory effects of drugs on Ca²⁺ uptake activities of isolated mitochondria. Inhibition 100 or 0% was defined as the mean of vehicle-treated samples or 100 μM CaCl₂-treated samples, respectively. Data obtained from 3 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test).

Figure 4

DS44170716 inhibits serum-induced mitochondrial Ca²⁺ influx in human HEK293A cells. (a) Representative temporal profiles of FCCP effect on serum-induced mitochondrial Ca²⁺ influx in human HEK 293 A cells. (b) FCCP effect on serum-induced mitochondrial Ca²⁺ influx in human HEK 293 A cells. The data shown were total mitochondrial Ca²⁺ influx from 0 to 17 seconds after the serum treatment. (c) Inhibitory effect of FCCP on serum-induced mitochondrial Ca²⁺ influx in human HEK 293 A cells. (d) Representative temporal profiles of DS44170716 effect on serum-induced mitochondrial Ca²⁺ influx in human HEK 293 A cells. (e) DS44170716 effect on serum-induced mitochondrial Ca²⁺ influx in human HEK 293 A cells. (f) Inhibitory effect of DS44170716 on serum-induced mitochondrial Ca²⁺ influx in human HEK 293 A cells. Data obtained from 3 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test).

Figure 5

DS44170716 decreases mitochondrial membrane potential in isolated mitochondria from pig heart. Relative membrane potential 0% or 100% was defined as 3 μM FCCP data or vehicle data, respectively. Data obtained from 4 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test).

Figure 6

Screening of mitochondrial drugs affecting Ca²⁺ uptake and membrane potential. (a) Effect of mitochondrial drugs on Ca²⁺ uptake activities in isolated mitochondria from pig heart. Data obtained from 3 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test). (b) Effect of mitochondrial drugs on membrane potential in isolated mitochondria from pig heart. Data obtained from 4 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to minimum dose group (Dunnett’s test).

Figure 7

DS44170716 inhibits enzyme activities of respiratory chain complexes. (a) Effect of DS44170716 on enzyme activities of mitochondrial complex III. (b) Effect of DS44170716 on enzyme activities of mitochondrial complex IV. (c) Effect of DS44170716 on enzyme activities of mitochondrial complex V. Inhibition 0% or 100% was defined as the mean of vehicle-treated samples or samples that were maximally inhibited by the positive control. Data obtained from 3 independent samples are shown as the mean with SEM. Asterisk shows P < 0.05 compared to vehicle group (Dunnett’s test).

Figure 8

Action mechanism of DS44170716. Mitochondrial respiratory chain complexes generate proton gradient, which is a driving force for Ca²⁺ uptake. Ca²⁺ overload into the matrix induces mitochondrial permeability transition (MPT). DS44170716 strongly inhibits complex III and weakly inhibits complex IV and V to decrease the potential. The decrease inhibits mitochondrial Ca²⁺ uptake and MPT. RuR or Ru360 blocks Ca²⁺ uptake by acting on MCU. CsA or NIM-811 inhibits PPIF, a central player promoting MPT.