Antagonism Smoothened leads to OFC

The activity of SMO is controlled by ligand binding (Kobilka 2007). Two separate binding pockets, one in the groove of the extracellular CRD and the other in the helices of the TMD have been identified (Nachtergaele, Mydock et al. 2012, Rana, Carroll et al. 2013, Wang, Wu et al. 2013, Byrne, Sircar et al. 2016, Huang, Zheng et al. 2018). These two binding pockets have been shown to interact in an allosteric manner (Nachtergaele, Mydock et al. 2012). The binding pocket in the helices of the TMD binds several SMO agonists including SAG as well as antagonists Vismodegib and Sonidegib. The CRD binding pocket binds cholesterol and its’ oxidized derivates (Byrne, Luchetti et al. 2018). The antagonist cyclopamine binds to the TMD binding pocket and inhibits SHH signal transduction. However, in mSMO carrying the mutations D477G/E552K that disable the TMD binding pocket, cyclopamine binds to the CRD pocket and activates the pathway (Huang, Nedelcu et al. 2016). To date several oxysterols including 20(S)-hydroxylcholesterol, 22(S)-hydroxylcholesterol, 7-keto-25-hydroxylcholesterol and 7-keto-27-hydroxylcholesterol have been identified as activators of SMO (Dwyer, Sever et al. 2007, Nachtergaele, Mydock et al. 2012, Myers, Sever et al. 2013). A binding site for 24(S),25-epoxycholesterol has been identified in the TMD pocket using cryo-EM of SMO in complex with 24(S),25-epoxycholesterol (Qi, Liu et al. 2019).

While it is well understood that cyclopamine is an antagonist of SMO, contradictory in vivo data was found regarding whether cyclopamine blocks SMO relocation to the primary cilia. Rohatgi et al used NIH 3T3s cell and found that cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10um (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009).  Corbit et al used a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005). Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.

The data presented in support of this KER includes in vivo studies that link antagonism of SMO with OFCs. In vitro data is presented in support of cyclopamine binding to SMO. The quantitative understanding of this relationship is low. No studies were found to exist to address dose response or time-scale data. Further work is needed to address these questions and create a better understanding of this relationship.

Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016). No data was found on how fast antagonism of SMO will stop its’ relocation to the primary cilia. Further work is needed to increase the understanding of this relationship and its’ time scale

Further work is needed to increase the understanding of this relationship and shed light on what other feedback/forward loops are at play.