Semisynthetic studies identify mitochondria poisons from botanical dietary supplements--geranyloxycoumarins from Aegle marmelos

Abstract

Bioassay-guided isolation and subsequent structure elucidation of a Bael tree Aegle marmelos lipid extract yielded two unstable acylated geranyloxycoumarin mixtures (1-2), six geranyloxycoumarins (3-8), (+)-9'-isovaleroxylariciresinol (9), and dehydromarmeline (10). In a T47D cell-based reporter assay, 1 and 2 potently inhibited hypoxia-induced HIF-1 activation (IC50 values 0.18 and 1.10 μgmL(-1), respectively). Insufficient material and chemical instability prevented full delineation of the fatty acyl side chain olefin substitution patterns in 1 and 2. Therefore, five fatty acyl geranyloxycoumarin ester derivatives (11-15) were prepared from marmin (3) and commercial fatty acyl chlorides by semisynthesis. The unsaturated C-6' linoleic acid ester derivative 14 that was structurally most similar to 1 and 2, inhibited HIF-1 activation with comparable potency (IC50 0.92 μM). The octanoyl (11) and undecanoyl (12) ester derivatives also suppressed HIF-1 activation (IC50 values 3.1 and 0.87 μM, respectively). Mechanistic studies revealed that these geranyloxycoumarin derivatives disrupt mitochondrial respiration, primarily at complex I. Thus, these compounds may inhibit HIF-1 activation by suppressing mitochondria-mediated hypoxic signaling. One surprising observation was that, while less potent, the purported cancer chemopreventive agent auraptene (8) was found to act as a mitochondrial poison that disrupts HIF-1 signaling in tumors.

Figure 1. Acylated geranyloxycoumarins 1 and 2 inhibit hypoxia-induced HIF-1 activation

T47D cells transfected with the pHRE-luc construct were exposed to hypoxia (1% O₂, 16 h, solid bar) and chemical hypoxia (10 μM 1,10-phenanthroline, 16 h, open bar) in the presence of 1 (A) and 2 (B) at the specified concentrations. The protein translation inhibitor cycloheximide (CHX, 10 μM) was used as a positive control. Data are presented as “% Inhibition” of the induced control (average + standard deviation, n = 3, representative of two independent experiments).

Figure 2. Effects of natural and semisynthetic geranyloxycoumarin derivatives on cellular respiration

A) Compounds 3–15 were evaluated at the concentrations of 3 μM (black bar), 10 μM (open bar), and 30 μM (striped bar) for their effects on cellular respiration in T47D cells. Rotenone (rot) was included as a positive control at the concentrations of 1 nM (black bar), 10 nM (open bar), and 100 nM (striped bar). Data shown are “% Inhibition” of the untreated control (average + deviation from average of two independent experiments). B) T47D cells were pretreated with rotenone (0.01 μM) and 14 (10 μM) for 2 h, the cells detached, and the rate of oxygen consumption monitored upon the addition of specified substrates and inhibitors. Changes in the slopes following substrate and/or inhibitor addition reflect alterations in the observed cellular respiration rates. C) Similar to those described in B), except that succinate was added to initiate respiration at complex II. D) Rates of oxygen consumption initiated by a mixture of malate/pyruvate (malate/pyruvate) and succinate (succinate) in T47D cells pretreated for 2 h with rotenone (0.01 μM, rot), 5 (10 μM), 11 (10 μM), and 14 (10 μM). Data shown are normalized to the rate of the untreated control (con).

Figure 3. Compounds 6, 8, and 10 inhibit mitochondrial respiration at ETC complex I

A panel of substrates and inhibitors were added to digitonin-permeabilized T47D cells in a sequential manner and the rates of oxygen consumption monitored. Changes in the slopes following substrate and/or inhibitor addition reflect alterations in the observed cellular respiration rates. Compounds 6 (30 μM, A), 8 (30 μM, B), and 10 (30 μM, C) failed to suppress mitochondrial respiration in the presence of succinate. All three compounds decreased mitochondrial respiration initiated by a mixture of malate/pyruvate at complex I (6, D; 8, E; and 10, F; respectively).

Figure 4. Concentration-response of 3–15 on breast tumor cell proliferation/viability

Cultured T47D (48 h, A; 144 h, C) and MDA-MB-231 (48 h, B; 144 h, D) cells were exposed to 3–15 at the concentrations of 1 μM (open bar), 3 μM (striped bar), 10 μM (gray bar), and 30 μM (black bar) for 48 h and 144 h (6 d). At the end of treatment, cell viability was determined by the sulforhodamine B method and presented as % Inhibition of the untreated control. For the 6 d exposure study, conditioned medium were replaced with fresh medium with compounds after 3 days. Cycloheximide (CHX, 10 μM) and rotenone (Rot; 1 nM, open bar; 10 nM, striped bar) were included as positive controls. Data shown are average + standard deviation (n = 3).