Fig 1.
Chemical structures of englerin A and englerin B.
Fig 2.
Englerin A affects proliferation of a subset of cancer cell lines across many cell lineages while englerin B is inactive.
(A) Scatterplot of englerin A cell line profiling experiment. (B) Scatterplot of englerin B cell line profiling experiment. Each point represents effect of englerin A or B on growth of a single tumor cell line. Y-axis indicates maximal effect on growth and X-axis indicates potency. Tumor cell line lineage is indicated by color and the legend is in the figure. Englerin A sensitive (circles), englerin A refractory (squares) and englerin A intermediate (diamonds) cell line calls are indicated.
Table 1.
Englerin A sensitivity calls across different tumor cell lineages.
Fig 3.
Sensitivity to englerin A induced growth inhibition correlates with expression of TRPC4.
(A) Volcano plot of gene expression features. X- axis indicates fold change of feature in sensitive versus refractory cell lines and Y-axis indicates statistical significance. (B) Waterfall plot showing TRPC4 expression levels (affymetrix microarray units) in englerin A sensitive (red) and refractory (blue) tumor cell lines.
Fig 4.
TRPC4 expression is necessary and sufficient for cell proliferation effects of englerin A.
(A) Effect of TRPC4 siRNA knockdown on viability of A-498 cells in the presence of englerin A. An siRNA targeting luciferase was used as a control (mean+/- S.E.M.) Percent reduction of the TRPC4 mRNA levels are indicated in the legend, KD stands for knockdown. TRPC4 mRNA levels were normalized to peptidyl prolyl isomerase A (PPIA) mRNA levels. (B) Effect of TRPC4 siRNA knockdown on viability of A-673 cells in the presence of englerin A (mean +/- S.E.M.) (C) Effect of overexpression of TRPC4 by transient transfection on viability of HEK293T cells in the presence of englerin A. TRPC4 expression vector concentrations are indicated by different shapes (D) Effect of TRPC4 expression on cell viability in the presence of an englerin A in HEK293T cells engineered to express TRPC4 under control of a Doxycycline (Dox) regulated promoter (mean +/- standard deviation). 100 ng/ml Dox (black circles), 0 ng/ml Dox (open circles). (E) Western blot visualizing the levels of TRPC4 in the presence or absence of 100 ng/ml Dox. (F) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-498 cells. (G) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-673 cells.
Fig 5.
Overexpression of TRPC5 can also confer sensitivity to englerin A.
(A) Expression of TRPC4 and TRPC5 in the cell lines from the CLiP experiment. Each circle represents a single cell line and expression levels were measured by microarray (TRPC4 probe 220818_s_at, TRPC5 probe 220552_at). The circle representing the DMS-79 cell line is indicated. (B) The effect of englerin A on cell viability in HEK293T cells transiently transfected with a vector expressing TRPC5 (mean +/- standard deviation).
Fig 6.
Englerin A agonizes the TRPC4/C5 ion channels and channel activation is needed for cell growth inhibition.
(A) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). (B) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. (C) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. (D) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) (E) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).
Table 2.
Englerin A selectivity against TRP family and non-TRP family ion channels.
Fig 7.
Serum stability and pharmacokinetics of englerin A.
(A) Levels of englerin A detected after incubation in serum from dog (open square), human (black square), rat (open circle), and mouse (black circle). (B) Pharmacokinetics of englerin A in rats after a single oral dose of 5 mg/kg (mean +/- S.E.M.), englerin A (black circle) and englerin B (open square).
Table 3.
Summary of pharmacokinetic experiments performed with englerin A.
Table 4.
Summary of tolerability experiments done in nude mice on englerin A.
Fig 8.
Effects of englerin A and englerin B on mean arterial pressure in anesthetized rats.
(A) Mean arterial blood pressure of anesthetized rats dosed with englerin A (mean +/- S.E.M.). (B) Mean arterial blood pressure of anesthetized rats dosed with englerin B (mean +/- S.E.M.).
Table 5.
Englerin A and B plasma levels and effects on animal health in anesthetized rat experiments.