Search for Hsp90 inhibitors with potential anticancer activity: isolation and SAR studies of radicicol and monocillin I from two plant-associated fungi of the Sonoran desert

Abstract

In an effort to discover small molecule inhibitors of Hsp90, we have screened over 500 EtOAc extracts of Sonoran desert plant-associated fungi using a two-stage strategy consisting of a primary cell-based heat shock induction assay (HSIA) followed by a secondary biochemical luciferase refolding assay (LRA). Bioassay-guided fractionation of extracts active in these assays derived from Chaetomium chiversii and Paraphaeosphaeria quadriseptata furnished the Hsp90 inhibitors radicicol (1) and monocillin I (2), respectively. In SAR studies, 1, 2, and their analogues, 3-16, were evaluated in these assays, and the antiproliferative activity of compounds active in both assays was determined using the breast cancer cell line MCF-7. Radicicol and monocillin I were also evaluated in a solid-phase competition assay for their ability to bind Hsp90 and to deplete cellular levels of two known Hsp90 client proteins with relevance to breast cancer, estrogen receptor (ER), and the type 1 insulin-like growth factor receptor (IGF-1R). Some inferences on SAR were made considering the crystal structure of the N-terminus of yeast Hsp90 bound to 1 and the observed biological activities of 1-16. Isolation of radicicol and monocillin I in this study provides evidence that we have developed an effective strategy for discovering natural product-based Hsp90 inhibitors with potential anticancer activity.

Figure 1

Cell-based heat shock induction assay (HSIA). The materials tested were DMSO (negative control), geldanamycin (GDA, positive control), EtOAc extracts, and major fractions, radicicol (1) and monocillin (2), derived from C. chiversii and P. quadriseptata. All samples except F29 were tested at 5 μg/mL; the concentration of F29 used was 2.5 μg/mL. The mean and standard deviation (SD) of triplicate determinations are presented, expressed as a percentage of the negative control; results are representative of three independent experiments.

Figure 2

Cell-based heat shock induction assay (HSIA) of compounds 1–16 at 10.0 and 5.0 μM, GDA (positive control) at 5.0 μM, and DMSO (negative control). Reporter activity in wells treated with test compounds relative to DMSO control is depicted. × indicates that no living cells were found in the wells treated with these compounds at 10 μM. The mean and SD of triplicate determinations are presented, expressed as a percentage of the negative control; results are representative of three independent experiments. An asterisk (*) indicates significant statistical difference of DMSO and the test condition (p value of <0.05) in a pooled-variance two-sample T-test.

Figure 3

(A) Inhibition of heat-denatured luciferase renaturation (luciferase-refolding assay; LRA) of compounds 1–16, GDA (positive control), and DMSO (negative control) after 3 min incubation at 28 °C. The mean and SD of triplicate determinations are presented, expressed as a percentage of the negative control; results are representative of three independent experiments. An asterisk (*) indicates significant statistical difference of DMSO and the test condition (p value of <0.05) in a pooled-variance two-sample T-test. (B) Time course of inhibition of renaturation of heat-denatured luciferase by radicicol [RAD (1)] at 1.0 μM and monocillin I [MON (2)] at 1.0 and 10.0 μM. The mean and SD of triplicate determinations are presented; results are representative of three independent experiments. Analysis of variance (ANOVA), analysis of covariance (ANCOVA), and standard post hoc tests (e.g., Tukey test) were performed on these data. ANCOVAs demonstrated significant differences between the curves of DMSO and the other test conditions. Post hoc tests showed at time zero there were no differences among the groups. At 3 min, DMSO was significantly different from the other compounds (p < 0.0001); MON at 1 μM and at 10 μM were not significantly different from one another (p = 0.088). At 30 min, DMSO was significantly different from the other compounds (p < 0.0001); MON at 1 μM also was significantly different from MON at 10 μM (p < 0.0001). At 60 min, DMSO was significantly different from the other compounds (p < 0.0001); MON at 1 μM also was significantly different from MON at 10 μM (p < 0.0001).

Figure 4

(A) GDA-immobilized bead competition assay of radicicol (1) and monocillin I (2). Soluble radicicol [RAD (1)] and monocillin I [MON (2)] compete for the binding of Hsp90 in cell lysate to the solid phase-immobilized derivative of GDA. (B) Immunoblot demonstrating depletion of the Hsp90 client protein estrogen receptor (ER) and type 1 insulin-like growth factor receptor (IGF-1R), and induction of the expression of Hsp70 by RAD (1) and MON (2). Results are representative of three independent experiments. DMSO lane B was loaded with half the amount of the total protein of lane A.

Figure 5

Partial structure–activity relationships for radicicol (1).