Small-molecule modulators of the Sonic Hedgehog signaling pathway
Shh pathway inhibition with non-naturally occurring small molecules
Cell-based assays with reporter gene constructs have been widely used for discovering non-naturally occurring small-molecule antagonists and agonists of Shh signaling. Sasaki et al. reported a construct with eight adjacent Gli binding sites upstream of a ﬁreﬂy luciferase reporter gene, which has been successfully used in cell lines, including the C3H10T1/2 and NIH3T3 (Shh-LIGHT2), to screen for small-molecule inhibitors of Gli activity. Among the Shh-pathway antagonists identiﬁed using this construct are Cur-61414, SANT1, SANT2, SANT3, SANT4, GANT61 and GANT58.
GANT58 and GANT61 act downstream of Smo. These two compounds have EC50 values of approximately 5 mMin Shh-LIGHT2 cells. When tested in C3H10T1/2 cells in the presence of Shh, GANT61 and GANT58 were able to repress alkaline phosphatase activity associated with Shh pathway activity. In Ptch1-/- MEFs, which lack Ptch1 function, the compounds retained inhibitory activity, indicating that their respective targets were downstream of Ptch1. When the two inhibitors were tested in MEF cells lacking both alleles of the endogenous downstream inhibitor Sufu, the compounds retained activity, whereas cyclopamine did not, as measured by Gli1 and Hhip1 mRNA levels. Thus, GANT58 and GANT61 act downstream of both Smo and Sufu.
Robotnikinin is a synthetic small-molecule inhibitor of Shh signaling that acts upstream of Smo. To date, it is the only small-molecule inhibitor of Shh signaling that acts upstream of Smo. Robotnikinin inhibits Gli transcription in Shh-LIGHT2 cells stimulated with ShhN, but when the Smo agonists purmorphamine or SAG were co-administered, its inhibitory activity was abolished. The compound is therefore able to inhibit Gli transcription by targeting a protein upstream of Smo in the Shh signaling pathway in Shh-LIGHT2 cells. Robotnikinin reduced alkaline phosphatase activity in a concentration-dependent fashion in C3H10T1/2 cells stimulated with ShhN, but this trend was eliminated when purmorphamine was co-administered. Thus, the activity of robotnikinin is not dependent on a cell line or reporter construct. Robotnikinin was also tested in primary human-derived keratinocytes stimulated with Shh. After 30 hours of treatment, Gli1 and Gli2 mRNA levels were reduced in a dose-dependent fashion, but this inhibition was eliminated with co-treatment with purmorphamine or SAG. Robotnikinin is able to inhibit Shh pathway activity in both mouse and human-derived cells upstream of Smo. In a Ptch1-/- MEF cell line with both Ptch1 alleles replaced with a LacZ reporter gene, no pathway inhibition was observed using robotnikinin. These data were consistent with a model where the small molecule inhibits the Shh pathway upstream of Ptch1. The compound also binds puriﬁed ShhN by SPR. These data provide strong evidence that robotnikinin directly targets the ShhN protein.
Shh pathway agonists: naturally occurring products
Cholesterol and other certain oxysterols participate in the activation of the Shh signaling pathway.
Beachy and co-workers reported that when intracellular sterol levels were depleted, but still remained suﬃcient for Shh post-translational modiﬁcation, cells became less responsive to ShhN.
Corcoran and Scott co-workers demonstrated that cholesterol, 20α-hydroxycholesterol, 22(S)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol activated Shh pathway activity, as measured by Ptch1–LacZ reporter activity in PZp53MED cells, while 7b-hydroxycholesterol had no eﬀect. In another study, 20α-hydroxy-cholesterol and 22(S)-hydroxycholesterol were found to induce Shh target gene upregulation as measured by rtPCR for Gli1 and Ptch1 in M2-10B4 cells. Cholesterol depletion therefore aﬀects Shh signaling directly, not merely through inhibition of autoprocessing. The exact mechanism of action of oxysterol Shh pathway agonists remains unknown, despite investigation. 20α-Hydroxycholesterol and 22(S)-hydroxycholesterol incubated with HEK293 cells overexpressing Smo did not compete with boron-dipyrromethene (BODIPY)–cyclopamine for Smo binding. A 1 : 1 combination of 20a-hydroxycholesterol and 22(S)-hydroxycholesterol (SS) was not able to shift the EC50 for cyclopamine in Ptch1 / MEFs, while Smo targeting agonists like SAG and purmorphamine can increase the cyclopamine’s EC50 dramatically. Furthermore, Smo-/- MEFs were not sensitive to SS, but cyclopamine was able to override the eﬀect of SS in C3H10T1/2 cells. Although the oxysterols do not bind directly to Smo, they still may indirectly aﬀect Smo, perhaps by stabilizing it in a conformation where it is less sensitive to Ptch1-mediated repression.
Benjamin Z. Stanton, Lee F. Peng*, Mol. BioSyst., 2010, 6, 44–54