A Hh-signaling agonist identified in a cell-based small-molecule screen. (a) A luciferase-based reporter assay of Hh signaling, showing a dose-response curve for the following: Hh protein (Hh); the small-molecule agonist Hh-Ag 1.1; Hh-Ag 1.1 in the presence of 0.3 nM Hh protein (Hh-Ag 1.1 + low Hh); or 0.3 nM Hh protein alone (low Hh). Data points represent the averages (n = 4) with standard deviations less than 15%. (b) The structure of Hh-Ag 1.1. (c) The output of a quantitative PCR analysis of Ptc1 and Gli1 mRNA levels from C3H10T1/2 cells exposed for 18 hours to an increasing dose of Hh-Ag 1.1. Data are graphed as relative activation versus Hh-Ag 1.1 concentration (μM). The 0 to 100% range was set using data from cells treated with 0 or 25 nM Hh protein; fold inductions for levels of Ptc1 and Gli1 mRNA were determined using GAPDH mRNA levels as internal standards. Each data point represents an average (n = 4) with standard deviation shown by error bars. (d) A luciferase-based reporter assay of Hh signaling showing dose-response curves (with concentrations in nM) for Hh protein and the five agonist compounds Hh-Ag 1.1, 1.2, 1.3, 1.4 and 1.5. Graphs are representative of multiple assays of these compounds. Data points represent the averages (n = 2) with standard deviations less than 15%. (e) Structures of Hh-agonist derivatives; 1.2 is a methylated analog, and 1.3 a methylated analog with a para-pyridyl moiety. (f) A proliferation assay of Hh-responsive primary neuronal precursors from postnatal day 4 rat cerebellum. [3H]-thymidine incorporation was measured 24 hours after the addition of the vehicle dimethyl sulfoxide ('vehicle'), Hh protein, or agonist. Hh protein was tested at 50 nM; Hh-Ag 1.1 was added at 5 and 1.75 μM; Hh-Ag 1.2 was added at 300 and 100 nM. Data points represent the averages (n = 4) with standard deviations depicted with error bars.