Purpose: With the advent of molecular medicine, the role of mitochondria has been expanded from the generation of metabolic energy to that of a multifunctional cellular hub. Consequently, mitochondria have become of great interest to explore novel treatment opportunities for many common diseases. Besides a treasure chest of novel therapeutic targets, mitochondria contain an increasing number of off-targets associated with side effects of many commonly used drugs. Drugs must pass through the largely impermeable mitochondrial inner membrane to reach their mitochondrial target, thus requiring a protein-mediated transport step. In addition, drugs can inhibit the physiological substrate transport, which could induce mitochondrial dysfunction. Strategies used to study drug mitochondrial transporter interactions are highly diverse and not straightforward. Therefore, we aimed to develop a blueprint strategy for the identification of drug effects on mitochondrial transporters. To date, most drugs, known to interfere with mitochondrial transporters, inhibit the ADP/ATP carrier (AAC), which imports ADP into the mitochondria in exchange for ATP. Hence, we used this carrier to provide the proof-of-principle of our strategy.
Methods: AAC-dependent 14C-ADP uptake was measured in Lactococcus Lactis whole cells overexpressing the human AAC isoform 1 (hAAC1), with or without a 15 min pre-incubation with 20 drugs previously reported to inhibit AAC function. Uptake was allowed to take place for 15 minutes, and of each drug four different concentrations (i.e. 100 uM, 10 uM, 1 uM, and 100 nM) were evaluated. Using an identical setup, 14C-ADP uptake was also measured in fusions with liposomes. To assess direct interaction of drugs with AAC we purified yeast AAC and evaluated the effects of all 20 drugs (100 uM) on thermal stability of the protein, which is known to increase upon inhibitor binding.
Results: Using the whole cell assay we could, however, only detect inhibition of ADP uptake with 4 out of 20 drugs (i.e. chebulinic acid, CD-437, equisetin, and tretinoin) at the highest concentration evaluated. The sensitivity could though be largely increased using L. Lactis membrane fusions, showing inhibition by 11 drugs. Moderate inhibition was observed with sertraline, furosemide, tretinoin, and quercetin decreasing ADP uptake rates by 12.5 ± 1.3 up to 40.9 ± 13.2%, whereas strong inhibition was observed with anthralin, CD-437, chebulinic acid, equisetin, nimesulide, and suramin decreasing ADP uptake by 54.1 ± 3.6 up to 72.6 ± 1.2%, which is comparable to the canonical AAC inhibitor carboxyatractyloside (59.9 ± 1.3%). Subsequent evaluation of the dose-dependency of these effects demonstrated a potent inhibitory effect of all six drugs, where the lowest IC50 value was observed with nimesulide (5.0 uM, 95%-CI 1.0-25.6). Finally, direct interaction with AAC was determined using a thermal stability assay with purified yeast AAC, in which only suramin induced a significantly increased thermostability (12.86 ± 0.01 degrees Celsius).
Conclusion: So far, we could only observe inhibition of human AAC1 by 11 out of 20 drugs previously reported to inhibit this carrier, which indicates the need for more specific approaches to investigate the effects of drugs on mitochondrial transport. This notion of large intra-assay variability is also confirmed by the differences we observed between whole cell and membrane fusion-based assays. More specific strategies are eventually expected to provide novel drug on- and off-targets, to add molecular relevance to known mitochondrial drug-targets, and to provide opportunities to increase mitochondrial targeting of drugs for clinical use further.
Martin King– Research Officer, University of Cambridge, Cambridge, England
Frans Russel– Professor, Radboud University Nijmegen Medical Centre, Nijmegen, Gelderland
Edmund Kunji– Groupleader, University of Cambridge, Cambridge, England