Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids

Abstract

Mitochondria have emerged as the major regulatory platform responsible for coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (cyt) c. As this oxidation occurs within the peroxidase complex of cyt c with CL, the latter represents a promising target for the discovery and design of drugs with anti-apoptotic mechanism of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogues of stearic acid TPP-n-ISA with different positions of the attached imidazole group on the fatty acid (n=6, 8, 10, 13 and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance, and electron spin echo envelope modulation) we demonstrated that TPP-n-ISA indeed were able to potently suppress CL induced structural re-arrangements in cyt c paving the way to its peroxidase competence. TPP-n-ISA analogues preserved the low spin hexa-coordinated heme iron state in cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of cyt c/CL complexes with a significant anti-apoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all atom molecular dynamics simulations. Based on the experimental data and computations predictions, we identified TPP-6-ISA as a candidate drug with optimized anti-apoptotic potency.

Inhibition of Peroxidase Activity of Cytochromec: De Novo Compound Discovery and Validation

“…In this conformer, the pocket enclosing the heme group is solvent-exposed and large enough to accommodate the binding of a drug-like molecule. As previously noted (Jiang et al, 2014), this conformation naturally tends to close down (into the native compact conformer) unless stabilized by a bound ligand. The ligand-binding properties of this site and the conformational space accessible upon ligand binding were characterized by two sets of runs (Table 1).…”

“…Screening of the PM against libraries of small molecules as well as a database of known drug-target pairs led to seven hits (drug-like compounds), in addition to the identification of three repurposable drugs. Biochemical experiments to inhibit cyt c peroxidase activity confirmed that seven of these 10 newly discovered compounds/drugs exhibit efficiencies comparable to or better than those observed (Jiang et al, 2014) for TPP-n-ISA molecules, opening the way to new molecular intervention strategies for modulating mitochondrial-mediated apoptosis.…”

“…This model helped us identify three Food and Drug Administration-approved (repurposable) drugs and seven novel compounds that inhibit the peroxidase activity of cyt c in a dosage-dependent manner. Four of the compounds were at least as effective as TPP-6-ISA, which we recently optimized for the inhibition of the peroxidase activity of cyt c using structurebased modeling (Jiang et al, 2014). Interestingly, compound L254614 [1-(4,9-dihydrothieno(2,3-c)(2)benzothiepin-4-yl)-1H-imidazole] exhibited remarkable potency, practically inhibiting complete cyt c peroxidase activity.…”

“…Using a combination of absorption spectroscopy and electron paramagnetic resonance measurements, along with molecular dynamics simulations, we showed that TPP-n-ISAs were able to suppress the CLinduced structural rearrangements in cyt c, which would otherwise give way to peroxidase activity (Jiang et al, 2014). Among these compounds, TPP-6-ISA proved to be particularly effective in inhibiting the peroxidase function in mouse embryonic cells exposed to ionizing radiation (Jiang et al, 2014).…”

Mitochondrial dysfunction in cardiac aging

“…Some of these therapies attempt to decrease the oxidative stress in the mitochondrial environment [203], while others focus on structural components of mitochondria [204–208]. It can be argued that CR and inhibition of mTOR signaling can do both, and thus, these two interventions, described above, and may also be considered to be mitochondrial therapies.…”