Synthesis and Antineoplastic Evaluation of Mitochondrial Complex II (Succinate Dehydrogenase) Inhibitors Derived from Atpenin A5

Abstract

Mitochondrial complex II (CII) is an emerging target for numerous human diseases. Sixteen analogues of the CII inhibitor natural product atpenin A5 were prepared to evaluate the structure-activity relationship of the C5 pyridine side chain. The side chain ketone moiety was determined to be pharmacophoric, engendering a bioactive conformation. One analogue, 1-(2,4-dihydroxy-5,6-dimethoxypyridin-3-yl)hexan-1-one (16 c), was found to have a CII IC₅₀ value of 64 nm, to retain selectivity for CII over mitochondrial complex I (>156-fold), and to possess a ligand-lipophilicity efficiency (LLE) of 5.62, desirable metrics for a lead compound. This derivative and other highly potent CII inhibitors show potent and selective anti-proliferative activity in multiple human prostate cancer cell lines under both normoxia and hypoxia, acting to inhibit mitochondrial electron transport.

Keywords: anticancer agents; atpenin A5; mitochondria; mitochondrial complex II; succinate dehydrogenase.

Figure 1

Structures of known complex II inhibitors.

Figure 2

Modelling of selected AA5 derivatives in the ubiquinone binding site of porcine heart mitochondria complex II (PDB ID: 3AEE). A) Hypothetical binding interactions of CII inhibitor 16c. B) Overlay of AA5 (7) (turquoise) and 16c (gold) in active site of porcine heart mitochondria complex II. C) Hypothetical binding interactions of inactive compound 18. D) Hypothetical binding interactions of inactive compound 24 (gold), overlayed with AA5 (7) (turquoise). Red;oxygen, blue; nitrogen, white; hydrogen, gold; carbon. Green dotted lines represent hydrogen bond (greater opacity represents stronger bond).

Figure 3

Complex II inhibitors are potent anti-proliferative agents. A) Dose-response curve of effect of complex II inhibitor 16c on percentage cell viability of DU-145 prostate cancer cells. B) Dose-response curve of effect of complex II inhibitor 16c on percentage cell viability of PC3 prostate cancer cells. C) Dose-response curve of effect of complex II inhibitor 16c on percentage cell viability of 22Rv1 prostate cancer cells. D) Dose-response curve of effect of complex II inhibitor 16c on percentage cell viability of low tumorigenic HEK293 cells. E) Dose-response curve of the effect of structurally similar but inactive complex II inhibitor 24 on percentage cell viability of DU-145 prostate cancer cells. F) Dose-response curve of the effect of clinical chemotherapeutic enzalutamide on percentage cell viability of 22Rv1 prostate cancer cells after 72 hour incubation. Cell viability measured after 48-hour incubation of compound unless otherwise noted. Values are the mean ± S.D. of triplicate experiments.

Figure 4

Complex II inhibitors with greater potency convey greater cytotoxicity. A) Dose-response curve of the effect of complex II inhibitor 16k on percentage cell viability of 22Rv1 prostate cancer cells. B) Dose-response curve of effect of complex II inhibitor 16k on the percentage cell viability of low tumorigenic HEK293 cells. Cell viability measured after 48-hour incubation of compound. Values are the mean ± S.D. of triplicate experiments.

Figure 5

Cytotoxic effect of the clinical chemotherapeutic etoposide, complex II inhibitor 16c and inactive control 18 in PC3 prostate cancer cells under hypoxia. Cell death was measured with Annexin V and Sytox Green. Values are the mean ± S.D. for triplicate experiments. A one-way ANOVA analysis was used to compare statistical difference between etoposide and 16c at 1 μM concentrations, p = 0.0068.

Figure 6

Complex II inhibitor 16c blocks mitochondrial respiration and function in 22Rv1 prostate cancer cells. A) Oxygen consumption rate (OCR), B) basal respiration, C) ATP production and D) maximal respiration are reduced in a dose-dependent manner. Error bars represent mean ± S.D. n = 3.

Scheme 1

Synthesis of hydrocarbon side chain derivatives of atpenin A5.

Scheme 2

Synthesis of oxidation state derivatives of atpenin A5.

Scheme 3

Synthesis of a methyl ether derivative of atpenin A5.