Targeting the Oncoprotein Smoothened by Small Molecules: Focus on Novel Acylguanidine Derivatives as Potent Smoothened Inhibitors

Abstract

Hedgehog-GLI (HH) signaling was originally identified as a critical morphogenetic pathway in embryonic development. Since its discovery, a multitude of studies have reported that HH signaling also plays key roles in a variety of cancer types and in maintaining tumor-initiating cells. Smoothened (SMO) is the main transducer of HH signaling, and in the last few years, it has emerged as a promising therapeutic target for anticancer therapy. Although vismodegib and sonidegib have demonstrated effectiveness for the treatment of basal cell carcinoma (BCC), their clinical use has been hampered by severe side effects, low selectivity against cancer stem cells, and the onset of mutation-driven drug resistance. Moreover, SMO antagonists are not effective in cancers where HH activation is due to mutations of pathway components downstream of SMO, or in the case of noncanonical, SMO-independent activation of the GLI transcription factors, the final mediators of HH signaling. Here, we review the current and rapidly expanding field of SMO small-molecule inhibitors in experimental and clinical settings, focusing on a class of acylguanidine derivatives. We also discuss various aspects of SMO, including mechanisms of resistance to SMO antagonists.

Keywords: GLI; acylguanidine derivative; cancer; drug-resistance; hedgehog; missense mutations; small molecule inhibitors; smoothened; targeted therapy.

Figure 1

Overview of the Hedgehog-GLI pathway with SMO and GLI antagonists. In absence of HH ligands (A), PTCH1 inhibits SMO by preventing its entry into the primary cilium (PC). GLI2 and GLI3 proteins are sequestered in the cytoplasm by SUFU and phosphorylated by PKA, GSK3β, and CK1, which create binding sites for the E3 ubiquitin ligase β-TrCP (β-Transducin Repeat-Containing Protein). GLI3 and, to a lesser extent, GLI2 undergo partial proteasome degradation, leading to the formation of repressor forms (GLI3^R/2^R) that translocate into the nucleus where they inhibit the transcription of HH target genes. Upon HH ligand binding (B), PTCH1 is displaced from the PC, allowing accumulation and activation of SMO. Active SMO relieves SUFU-mediated suppression of GLI2 and GLI3 within the PC. GLI2 and GLI3 maintain their full-length status and bypass phosphorylation. Activator forms of GLI (GLI1^A/2^A/3^A) translocate into the nucleus, where they induce the transcription of HH pathway target genes. Movement of GLI2 and GLI3 within the PC occurs in conjunction with KIF7, a member of the kinesin family of anterograde motor proteins. SMO (orange box) and GLI (light blue box) antagonists are indicated in (B). GLI inhibitor Pyrvinium enhances CK1α-dependent degradation of GLI^A. CK1, casein kinase 1; GSK3β, glycogen synthase kinase 3β; PKA, protein kinase A; PTCH1, Patched 1; SMO, Smoothened; SUFU, Suppressor of Fused; HH, Hedgehog; KIF7, kinesin family member 7; ATO, arsenic trioxide.

Figure 2

Mechanisms of Hedgehog pathway activation in cancer. Ligand-independent activation is due to inactivating mutations in the negative regulators PTCH1 or SUFU, activating mutations in SMO, or amplification of GLI activators. Ligand-dependent activation occurs through autocrine, paracrine, or inverse paracrine mechanisms (see text for details). In the autocrine mechanism, tumor cells secrete and respond to HH ligands; in the paracrine pattern tumor cells produce HH ligands, which activate HH pathway in stroma cells; in the reverse-paracrine mechanism stroma cells produce HH ligands, which activate HH pathway in tumor cells. PTCH1, Patched 1; SMO, Smoothened; SUFU, Suppressor of Fused; HH, hedgehog ligand; GLI^A, GLI activators; IL6, Interleukin-6; IGF, Insulin Growth Factor; VEGF, Vascular Endothelial Growth Factor; Wnt, Wingless/Integrated.

Figure 3

Schematic structure of the human SMO protein, showing the location of oncogenic mutations (red), vismodegib-resistance mutations (green), sonidegib-resistance mutations (orange) and oncogenic mutations associated with vismodegib resistance (red, green and black). Numbers represent amino acids. Human SMO contains 787 amino acids organized in three main domains: the N-terminal extracellular domain (ECD) (residues 1–220), containing a cysteine-rich domain (CRD); the heptahelical membrane spanning (7-TM) domain (TMD) (residues 221–558); the C-terminal cytoplasmic domain (residues 559–787). The TMD consists of seven transmembrane domains connected by three extracellular loops (ECL1–3) outside of the plasma membrane and three intracellular loops (ICL1–3). See Table 1 for details about mutations.

Figure 4

Discovery, optimization, and biological characterization of acylguanidine and acylthiourea derivatives. A virtual screening identified MRT-10 (acylthiourea) and MRT-14 (acylurea analog) as novel SMO inhibitors. Further structure-activity relationship (SAR) study yielded to the acylguanidine MRT-83, which showed nanomolar antagonist potency towards SMO. Further elongation of the biaryl moiety of MRT-83 led to acylguanidine MRT-92 (Compound 1), with a phenylethylphenyl tail. Addition of a fluorine (F) atom or substitution of the NH group with a sulphur (S) atom in MRT-92 led, respectively, to Compound 2 and Compound 3 (MRT-95). All these compounds show antagonist activity with nanomolar potency, except for Compound 3 (MRT-95), which displays much reduced activity. Inhibition of GLI1 protein expression gives an indication of the degree of HH pathway inhibition.

Figure 5

Schematic representation of the mechanisms of resistance to SMO inhibitors. These are represented by: activating mutations in SMO (1); loss of SUFU (2); amplification of GLI2 gene (3); activation of the PI3K/AKT/mTOR pathway, which induces S6K1-dependent phosphorylation and activation of GLI1 (4); activation of the RAS/RAF/MEK/ERK pathway (5); phosphorylation- dependent activation of GLI1 by aPKCι/λ (6) or DYRK1B (7); histone deacetylases (8); BRD4 protein (9). SMO, Smoothened; SUFU, Suppressor of Fused; IGFR, Insulin growth factor receptor; PTEN, Phosphatase and tensin homolog; PI3K, Phosphatidylinositol-4,5-bisphosphate 3-kinase; mTOR, mammalian target of Rapamycin; GSK3β, Glycogen synthase kinase 3β; S6K1, Ribosomal protein S6 kinase beta-1; EGFR, Epidermal growth factor receptor; PDGFR, Platelet-derived growth factor receptor; MEK1/2, MAP (Mitogen-activated protein) Kinase/ERK (Extracellular signal-Regulated Kinase) Kinase 1; HDAC, Histone deacetylase; BRD4, Bromodomain-containing protein 4.