The novel cyclophilin inhibitor C105SR reduces hepatic ischaemia-reperfusion injury via mitoprotection

Abstract

Background & aims: Mitochondrial permeability transition pore (mPTP) opening is critical for mediating cell death during hepatic ischaemia-reperfusion injury (IRI). Blocking mPTP opening by inhibiting cyclophilin D (CypD) is a promising pharmacological approach for the treatment of IRI. Here, we show that diastereoisomers of a new class of small-molecule cyclophilin inhibitors (SMCypIs) have properties that make them attractive candidates for the development of therapeutic agents against liver IRI.

Methods: Derivatives of the parent SMCypI were synthesised and evaluated for their ability to inhibit CypD peptidyl-prolyl cis-trans isomerase (PPIase) activity and for their mitoprotective properties, evaluated by measuring mitochondrial swelling and calcium retention capacity in liver mitochondria. The ability of the selected compounds to inhibit mPTP opening was evaluated in cells subjected to hypoxia/reoxygenation using a calcein/cobalt assay. Their ability to inhibit cell death was evaluated in cells subjected to hypoxia/reoxygenation by measuring lactate dehydrogenase (LDH) release, propidium iodide staining, and cell viability. The compound performing best in vitro was selected for in vivo efficacy evaluation in a mouse model of hepatic IRI.

Results: The two compounds that showed the strongest inhibition of CypD PPIase activity and mPTP opening, C105 and C110, were selected. Their SR diastereoisomers carried the activity of the racemic mixture and exhibited mitoprotective properties superior to those of the known macrocyclic cyclophilin inhibitors cyclosporin A and alisporivir. C105SR was more potent than C110SR in inhibiting mPTP opening and prevented cell death in a model of hypoxia/reoxygenation. Finally, C105SR substantially protected against hepatic IRI in vivo by reducing hepatocyte necrosis and apoptosis.

Conclusions: We identified a novel cyclophilin inhibitor with strong mitoprotective properties both in vitro and in vivo that represents a promising candidate for cellular protection in hepatic IRI.

Impact and implications: Hepatic ischaemia-reperfusion injury (IRI) is one of the main causes of morbidity and mortality during or after liver surgery. However, no effective therapies are available to prevent or treat this devastating syndrome. An attractive strategy to prevent hepatic IRI aims at reducing cell death by targeting mitochondrial permeability transition pore opening, a phenomenon regulated by cyclophilin D. Here, we identified a new small-molecule cyclophilin inhibitor, and demonstrated the enhanced mitoprotective and hepatoprotective properties of one of its diastereoisomers both in vitro and in vivo, making it an attractive lead compound for subsequent clinical development.

Keywords: Cellular protection; Liver necrosis; Mitochondrial calcium retention capacity; Mitochondrial permeability transition pore; Mitochondrial swelling; Peptidyl-prolyl cis-trans isomerase activity.

Graphical abstract

Fig. 1

Concentration-dependent enhancement of calcium retention capacity of isolated mouse liver mitochondria by SMCypI C31 derivatives. (A) Chemical structure of compound C31. (B) Surface representation of CypD showing the PPIase catalytic site (S1 pocket) and the gatekeeper pocket (S2 pocket). (C) CRC of mouse liver mitochondria in the absence (Ctrl) or in the presence of CsA (1 μM), ALV (1 μM), C31 (100 μM), or C31 derivatives (100 μM), ranked by decreasing level of CRC, expressed as nmol of calcium per mg of mitochondrial proteins. Data are shown as mean ± SEM. One way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F. ∗∗p <0.001 vs. Ctrl; ∗∗∗∗p <0.0001 vs. Ctrl; ^#p <0.05 vs. CsA; ^##p <0.01 vs. CsA; ^###p <0.0001 vs. CsA. ALV, alisporivir; CsA, cyclosporin A; CypD, cyclophilin D; CRC, calcium retention capacity; PPIase, peptidyl-prolyl cis-trans isomerase; SMCypIs, small-molecule cyclophilin inhibitors.

Fig. 2

Biological activity of the racemic mixtures and diastereoisomers of compounds C105 and C110. (A) Inhibition of CypD PPIase activity by C105 (left) and C110 (right) racemic mixtures and their diastereoisomers at 10 μM expressed as percent of the complete inhibition of CypD PPIase activity induced by CsA. (B) Concentration-response curves of CypD PPIase activity inhibition by diastereoisomers C105SR and C110SR. (C) Mitochondrial CRC of mouse liver mitochondria in the absence (Ctrl) or in the presence of C105 (left) and C110 (right) racemic mixture and their diastereoisomers at 100 μM. Data are shown as mean ± SEM. One way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F. ∗p <0.05 vs. Ctrl;; ∗∗∗p <0.001 vs. Ctrl; ∗∗∗∗p <0.0001 vs. Ctrl; ^#p <0.05 vs. racemic mixture. (D) Concentration-response curves of mitochondrial CRC of compounds C105SR and C110SR. (E) Mitochondrial CRC in the absence or in the presence of 1 μM CsA, or 1 μM ALV or increasing concentrations of C31, C105, and C105SR (left) or C31, C110, and C110SR (right). Data are shown as mean ± SEM. One way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F. ∗p <0.05 vs. Ctrl; ^#p <0.05 vs. racemic mixture; ^&p <0.05 vs. C31; ^£p <0.05 vs. CsA. CRC, calcium retention capacity; CsA, cyclosporin A; CypD, cyclophilin D; PPIase, peptidyl-prolyl cis-trans isomerase.

Fig. 3

In vitro inhibition of mPTP opening and reduction of necrosis by C105SR and C110SR in a model of hepatic hypoxia/reoxygenation. Cells were pretreated with 1 μM calcein and 1 mM CoCl₂ for 30 min and 10 min, respectively, then subjected to 4 h of hypoxia (1% O₂) followed by 1 h of reoxygenation (21% O₂) in the presence of 3 μM propidium iodide (PI). CsA and ALV were used as references. CsA, ALV, C31, C105SR, and C110SR were added at 1 μM for the entire duration of hypoxia/reoxygenation. (A) Representative images of calcein (green) and PI (red) labelling in cells exposed to normoxia (control) or hypoxia/reoxygenation in the absence (vehicle) or in the presence of CsA, ALV, C31, C105SR, or C110SR (original magnification, 400 × ). (B) Calcein fluorescence in cells exposed to normoxia (Ctrl) or hypoxia/reoxygenation in the absence (vehicle) or in the presence of CsA, ALV, C31, C105SR, or C110SR. Data are shown as mean ± SEM. One way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F. ^####p <0.001 vs. vehicle. (C) PI fluorescence in cells exposed to normoxia (Ctrl) or hypoxia/reoxygenation in the absence (vehicle) or in the presence of CsA, ALV, C31, C105SR or C110SR. Data are shown as mean ± SEM. One way ANOVA analysis followed by a Tukey’s or a Dunnett's post-test if ANOVA produced a significant value of F. ∗∗∗p <0.001 vs. Ctrl; ∗∗∗∗p <0.0001 vs. Ctrl; ^#p <0.05 vs. vehicle; ^##p <0.01 vs. vehicle; ^###p <0.001 vs. vehicle. ALV, alisporivir; CsA, cyclosporin A; mPTP, mitochondrial permeability transition pore.

Fig. 4

In vitro reduction of LDH release and increase in cell viability induced by C105SR and C110SR in a model of hepatic hypoxia/reoxygenation. Cells were subjected to 4 h of hypoxia (1% O₂) followed by 2 h of reoxygenation (21% O₂). CsA and ALV were used as references. CsA and ALV were added at 1 μM while C31, C105SR, and C110SR were added at increasing concentrations during the hypoxic and reoxygenation phases. (A) LDH release from cells exposed to normoxia (Ctrl) or hypoxia/reoxygenation in the absence (vehicle) or in the presence of CsA, ALV, or increasing concentrations of C31, C105SR, or C110SR expressed as percentage of LDH release in cells treated with a lysis buffer. Data are shown as mean ± SEM. One way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F. ∗∗∗p <0.001 vs. Ctrl; ^##p <0.01 vs. Vehicle. (B) Cell viability measured by MTT assay in cells exposed to normoxia (Ctrl) or hypoxia/reoxygenation in the absence (vehicle) or in the presence of CsA, ALV, or increasing concentrations of C31, C105SR, or C110SR expressed as percentage of control. Data are shown as mean ± SEM. One-way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F.∗∗∗p <0.001 vs. Ctrl; ^###p <0.001 vs. Vehicle; ^####p <0.0001 vs. Vehicle. (C) Caspase 3/7 activity expressed as percentage of control (Ctrl) in cells subjected to 4 h of hypoxia and 2 h of reoxygenation treated with vehicle or increasing concentrations of C105SR or C110SR. Data are shown as mean ± SEM. One way ANOVA analysis followed by Tukey’s or Dunnett's post-test if ANOVA produced a significant value of F ∗∗∗∗p <0.0001 vs. Ctrl; ^####p <0.0001 vs. Vehicle. ALV, alisporivir; CsA, cyclosporin A; LDH, lactate dehydrogenase.

Fig. 5

Docking of C31 and C105SR on CypD. CypD, C31, and C105SR are shown in grey, pink, and green, respectively. (A) Large view of C105SR docked on CypD. (B) Zoom view of C105SR docked on CypD showing amino acid interactions. (C) Chemical structures of C31 and C105SR, showing their similar backbones and the differences in R1, R2, and R3 functional regions. (D) Superposition of docking poses of C31 and C105SR on CypD. CypD, cyclophilin D.

Fig. 6

In vivo protective effects of C105SR (50 mg/kg) against hepatic ischaemia–reperfusion injury. (A) Representative haematoxylin and eosin staining images (magnification, 200 × ) of liver lobes (left) and percent of hepatocyte necrosis (right) in mice subjected to laparotomy without (sham vehicle) or with ischaemia–reperfusion (IR) in the absence (vehicle) or in the presence of C105SR. n = 8 for sham vehicle; n = 24 for IR vehicle; n = 13 for IR C105SR. Data are shown as mean ± SEM. Mann–Whitney U test ∗∗∗∗p <0.0001 vs. sham vehicle; ^####p <0.0001 vs. IR vehicle. (B) Serum ALT and AST levels in mice subjected to laparotomy without (sham vehicle) or with ischaemia–reperfusion (IR) in the absence (vehicle) or in the presence of C105SR. n = 8 for sham vehicle; n = 24 for IR vehicle; n = 13 for IR C105SR. Data are shown as mean ± SEM. Mann–Whitney U test. ∗∗∗∗p <0.0001 vs. sham vehicle; ^##p <0.01 vs. IR vehicle (C) Representative TUNEL staining (left) and number of TUNEL-positive cells (right) in livers of mice subjected to laparotomy without (sham vehicle) or with ischaemia–reperfusion (IR) in the absence (vehicle) or in the presence of C105SR. n = 4 for sham vehicle; n = 8 for IR vehicle; n = 8 for IR C105SR. Data are shown as mean ± SEM. Mann–Whitney U test ∗∗p <0.01 vs. sham vehicle; ∗∗∗p <0.001 vs. sham vehicle; ^##p <0.01 vs. IR vehicle. ALT, alanine aminotransferase; AST, aspartate aminotransferase; IR, ischaemia–reperfusion.