(-)-Englerin A is a potent and selective activator of TRPC4 and TRPC5 calcium channels

Abstract

Current therapies for common types of cancer such as renal cell cancer are often ineffective and unspecific, and novel pharmacological targets and approaches are in high demand. Here we show the unexpected possibility for the rapid and selective killing of renal cancer cells through activation of calcium-permeable nonselective transient receptor potential canonical (TRPC) calcium channels by the sesquiterpene (-)-englerin A. This compound was found to be a highly efficient, fast-acting, potent, selective, and direct stimulator of TRPC4 and TRPC5 channels. TRPC4/5 activation through a high-affinity extracellular (-)-englerin A binding site may open up novel opportunities for drug discovery aimed at renal cancer.

Keywords: antitumor agents; calcium ions; ion channels; natural products.

Figure 1. (–)EA is a potent and highly efficacious activator of TRPC4/5 channels.

(a-c, e-f) Measurements of the free intracellular calcium ion (Ca²⁺ _i) concentration shown as the fura-2 fluorescence (F) ratio or change (Δ) in this ratio. Extracellular Ca²⁺ was 1.5 mM unless otherwise indicated. (a) Genetically-modified HEK 293 cells not induced (HEK (Tet-)) or induced using tetracycline (Tet+) to over-express TRPC4 (HEK-TRPC4). After 1 min of recording 3 nM (–)EA was applied to the extracellular medium as indicated by the vertical dashed line (N=5 each). Representative of n=4. (b-e) Genetically-modified HEK 293 cells induced to over-express TRPC4 (HEK-TRPC4). (b) Extracellular application of 100 µM carbachol (CCh) and then, in addition, 100 nM (–)EA (N=6 each). Representative of n=3. (c) Application of 100 nM (–)EA in the presence (1.5 Ca²⁺ _e) or absence (0 Ca²⁺ _e) of extracellular 1.5 mM Ca²⁺ (N=6 each). Representative of n=3. (d) Whole-cell voltage-clamp recording of membrane current (I) from a single-cell during ramp changes in membrane voltage (V) from –100 to +100 mV, shown during the application of extracellular vehicle (dimethyl sulfoxide and pluronic acid) and then 100 nM (–)EA. The arrow points to the seat-like inflection in the I-V. Typical of n=12 (4 with standard pipette solution, 8 with aspartate solution). (e) Concentration-response data for (–)EA (n/N=4/18-19). The fitted curve is a Hill equation indicating 50 % maximum effect (EC₅₀) at 11.2 nM. (g) As for (e) except the cells were genetically-modified HEK 293 cells induced to over-express TRPM2 (HEK-TRPM2) (n/N=3/18). (g-i) Ionic currents across outside-out membrane patches from genetically-modified HEK 293 cells induced to over-express TRPC4 (HEK-TRPC4). (g) As indicated by the inset diagram 1 mM guanosine 5′-[β-thio]diphosphate (GDP-β-S) was in the patch pipette and (–)EA (EA) was bath-applied to the extracellular surface of the membrane (indicated by horizontal bars above the experimental traces). ML204 (ML) was also bath-applied. The vehicle (dimethyl sulfoxide and pluronic acid) was kept constant throughout the recording. Ramp changes in membrane voltage from –100 to +100 mV were applied every 10 s and the currents sampled at –100 and +100 mV are displayed. Typical of n=4. (h) As for (g) except, as indicated by the inset diagram, GDP-β-S (and ATP) were omitted from the patch pipette and the pipette contained 100 nM (–)EA. Typical of n=3. (i) From the experiment shown in (h), full current traces during two ramp changes in voltage, one before ((–)EA i) and the other after bath-application of (–)EA ((–)EA _e+i). The arrow points to the seat-like inflection in the I-V.

Figure 2. (–)EA activated endogenous TRPC4-containing channels of renal carcinoma cells are a mechanism for selective drug-induced cell death.

(a-d) Measurements of the free intracellular calcium ion (Ca²⁺ i) concentration in A498 cells shown as the fura-2 fluorescence (F) ratio or change (Δ) in this ratio. (a) Example effect of extracellular application of (–)EA or its vehicle control (n/N=1/5 each). (b) Concentration-response data (n/N=3/45) with a fitted Hill equation indicating an EC₅₀ of 9.5 nM. (c) Mean responses after 4 min exposure to vehicle, 1 µM (–)EA, or 1 µM (+)EA (n/N=4/23 each). (d) Mean responses after 4 min exposure to vehicle, 1 µM (–)EA, or 1 µM (–)EA in the presence of 5 µM ML204 (n/N=4/23 each). (e) Whole-cell voltage-clamp recording of membrane current from a single A498 cell during ramp changes in membrane voltage from -100 to +100 mV applied every 10 s. Only current sampled at –100 and +100 mV is displayed. 100 nM (–)EA and 5 µM ML204 were bath-applied as indicated by the horizontal bars. Representative from n=11 (standard pipette solution) and n=5 (aspartate pipette solution). (f) From the experiment shown in (e) full current traces during two ramp changes in voltage, one during the initial application of vehicle (veh.) (dimethyl sulfoxide and pluronic acid) and the other after the application of (–)EA and before ML204. (g, h) As for (e, f) except genetically-modified HEK 293 cells induced to over-express TRPC4 and transiently express TRPC1 (HEK C4+C1). Representative from n=3 (standard pipette solution).

Figure 3. TRPC4/5 are involved in the (-)EA-mediated decrease of cell viability

(a-b) HEK293 cells were transiently transfected with plasmids for ectopic expression of TRPC4 (a) or TRPC5 (b). 8 h later cell were replated and allowed to grow for 48 h. Cells were treated with different concentrations of (-)EA for 4 h prior to determination of cell viability using the WST-1 reagent. Data are mean values (N=4) ± s.d. and are normalized to cells treated with DMSO. Data were fitted using four-parameter Hill equation and are representative of three independent experiments. (c) Different concentrations of EGTA and 100 nM (-)EA were added to A498 cells prior to subsequent determination of cell viability. Data are shown as mean values ± s.d. (n=3, N=4). (d) A498 cells were incubated for 24 h in medium containing 180 µM CaCl₂ or 1.8 mM CaCl₂ prior to addition of different concentrations of (-)EA followed by determination of cell viability. Data are shown as mean values ± s.d. (n=3, N=4) and were fitted using four-parameter Hill equation indicating IC₅₀ of 77.7 nM in presence of 180 µM CaCl₂ and 7.5 nM in presence of 1.8 mM CaCl₂.