A small molecule that binds Hedgehog and blocks its signaling in human cells

Abstract

Small-molecule inhibition of extracellular proteins that activate membrane receptors has proven to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic hedgehog (Shh) protein and blocks Shh signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.

Figure 1. Characterization of SMM hit, 1

(a) Structure of 1. (b) SPR plot of 1 binding to purified ShhN. The plot shows normalized response units (RUs) on the y-axis and time (s) on the x-axis. The concentrations plotted are 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, and 25 μM, in order of increasing normalized RUs. (c) Luminescence plots for a Gli-dependent firefly luciferase reporter gene assay of 1 at the indicated concentrations. ShhN represents a positive control for medium containing ShhN palmitoylated at the N-terminus. The assays were performed at 0.25 % (v/v) DMSO. Each value represents the average of five experiments, with the error bar denoting the standard deviation.

Figure 2. Robotnikinin

(a) The structure of robotnikinin, which is the compound that resulted from follow-up chemistry efforts to optimize potency. (b) SPR curve of robotnikinin showing concentration-dependent binding to purified ShhN. Normalized RUs are plotted over a time course. The concentrations plotted are 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, and 25 μM, in order of increasing RUs (c) Inhibition of Gli signaling by robotnikinin in Shh-LIGHT2 cells stimulated with medium containing ShhN palmitoylated at the N-terminus, relative to 6.25 μM cyclopamine (a small-molecule inhibitor of Smoothened). Shh-LIGHT2 cells stimulated with N-palmitoylated ShhN along with 3.6 μM purmorphamine or 100 nM SAG (small-molecule activator of Smoothened) showed negligible inhibition at the indicated concentrations of inhibitor. (d) Robotnikinin lowers levels of endogenous Gli2 mRNA (analyzed by qPCR) in primary human keratinocytes in a dose-dependent manner; this effect is blocked by the co-administration of Smo agonists. Note that there is some Gli expression in the absence of exogenous Shh due to the presence of a basal amount of Shh in the growth medium. (e) When analyzed by qPCR, synthetic human skin displayed Gli1 and Gli2 transcriptional repression in the presence of varying concentrations of robotnikinin. (f) Robotnikinin inhibits the induction of the Shh pathway. Our experiments support a mechanism involving inhibition of the actions of Shh, either directly or indirectly by interfering with a precursor complex.