Identification of a novel toxicophore in anti-cancer chemotherapeutics that targets mitochondrial respiratory complex I
- MRC Toxicology Unit, University of Cambridge, United Kingdom
- School of Pharmacy, Biodiscovery Institute, University of Nottingham, United Kingdom
- MRC Mitochondrial Biology Unit, University of Cambridge, United Kingdom
Figures
Figure 1 with 1 supplement
Mubritinib does not inhibit HER2, but inhibits ATP production and beat rate of cardiomyocytes.
(A) Western blot analysis of the HER2-overexpressing cell line, BT474, treated with increasing doses of mubritinib. HER2 activity was assessed with antibodies against phosphorylated HER2 …
Figure 1—figure supplement 1
Mubritinib targets complex I in cardiomyocytes.
(A) Quantification of HER2 phosphorylation from Figure 1A displayed relative to the untreated sample. Error bars represent standard deviation (n = 3) and significance was assessed using unpaired …
Figure 2 with 2 supplements
Mubritinib is an inhibitor of mitochondrial complex I.
(A) Variants of mubritinib were synthesised with alterations to the trifluoromethylphenyl group (2, 3 and 4) or the triazole (5, 6, 7 and 8). Mubritinib (1) and mubritinib synthesised ‘in house’ …
Figure 2—figure supplement 1
Mubritinib inhibits OCR in H9c2 and hESC-CM.
(A and B) Representative seahorse trace (for Figure 2B) from (A) H9c2 or (B) hESC-CM cells in glucose containing media and treated with mubritinib (1 μM) or oligomycin A (1 μM).
Figure 2—figure supplement 2
Complex I inhibition by mubritinib and the synthesised variant compounds.
(A) Mubritinib and the compound variants (i-viii) were incubated with mitochondrial membranes at the concentrations shown. The rate of NADH oxidation was measured spectrophotometrically, and data …
Figure 3 with 3 supplements
The toxicophore present in carboxyamidotriazole inhibits mitochondrial complex I.
(A) Chemical structure of carboxyamidotriazole (CAI) (9) and three variants whereby the core triazole ring was replaced with either a pyrazole (10 and 11) or imidazole (12). For the pyrazoles, 11 is …
Figure 3—figure supplement 1
The toxicophore in CAI inhibits mitochondrial complex I.
(A and B) Representative seahorse trace (for Figure 3C) of H9c2 cells grown in glucose containing media and treated with either (A) 3 μM or (B) 10 μM of the indicated compounds. (C) A549 cells were …
Figure 3—figure supplement 2
Complex I inhibition by mubritinib and the synthesised variant compounds.
(A) CAI and the compound variants (i–v) were incubated with mitochondrial membranes at the concentrations shown. The rate of NADH oxidation was measured spectrophotometrically, and data were fit to …
Figure 3—figure supplement 3
CAI inhibits signalling pathways responsive to changes in energy status.
(A) Western blot analysis of A549 cells treated with 5 μM of CAI (9), 5 μM of 11 (inactive CAI derivative), 2.5 μM bepridil hydrochloride (non-specific calcium channel inhibitor) or 1 μM rotenone …
Figure 4 with 1 supplement
The toxicophore present in mubritinib and CAI is required for efficacy as an anti-cancer agent.
(A and B) HL60 cells grown in media containing galactose (A) or glucose (B) as an energy source treated with mubritinib (1) (2 μM), CAI (9) (5 μM) or the inactive derivatives 5 (2 μM) or 11 (5 μM) …
Figure 4—figure supplement 1
CAI and mubritinib inhibit cell growth and induce apoptosis in glycolytic-deficient tumour cell lines.
(A and B) HL60 cells grown in media containing galactose (A) or glucose (B) as an energy source treated with mubritinib (1) (2 μM), CAI (9) (5 μM) or the inactive derivatives 5 (2 μM) or 11 (5 μM). …
Figure 5
Schematic diagram to show the cell-wide effect of ETC complex I inhibition by the toxicophore.
Chemical inhibition of mitochondrial respiratory complex I with mubritinib and CAI leads to a decrease in cellular ATP and the subsequent activation of the energy sensor AMPK. Importantly, AMPK has …
Appendix 1—figure 1
Mubritinib analogues.
Appendix 1—figure 2
Carboxyamidotriazole analogues.
Additional files
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Supplementary file 1
Identification of compounds which contain a non-fused triazole in a conformation similar to mubritinib.
ChEMBL was searched for drugs containing a non-fused triazole, which led to the identification of a number of small molecules that are used clinically either routinely or in trials use e.g. the antibiotic tazobactam, the anti-epileptic drug rufinamide, and the cancer chemotherapeutic carboxyamidotriazole. These all contain the triazole ring, but have differing associated physicochemical properties.
- https://cdn.elifesciences.org/articles/55845/elife-55845-supp1-v2.xlsx
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Supplementary file 2
Ion channel binding assay.
The Ca2+ ion channel binding assay to test the activity of CAI (9), 10, 11, mubritinib (1) and 6 was performed by Eurofins. The percentage inhibition of ion channel was calculated relative to the positive control (1,4,5-IP3). On the scale used a score of 1 = no binding and a score of 100 = binding. The data show that there is no direct binding of these drugs to the ion channels.
- https://cdn.elifesciences.org/articles/55845/elife-55845-supp2-v2.docx
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Supplementary file 3
Ketoconazole, terconazole and rufinamide all contain a heterocyclic 1,3-nitrogen motif.
The compounds listed were incubated with mitochondrial membranes and the rate of NADH oxidation was measured spectrophotometrically.
- https://cdn.elifesciences.org/articles/55845/elife-55845-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55845/elife-55845-transrepform-v2.docx
