Naoaki Fujii

Hedgehog-Gli Signaling Pathway Inhibitors as Anticancer Agents

Cancer drug discovery has undergone a paradigm change over the past few years, from predominantly cytotoxic agent-based therapy to therapy aimed at genetic and molecular targets, thanks to a growing understanding of the genes and pathways responsible for cancer initiation and progression and to new drug discovery technologies. The success of drugs like trastuzumab, imatinib, gefitinib, and erlotinib has demonstrated that the targeting of specific oncogenic signal transduction pathways can be clinically useful.1 One such pathway, the Hedgehog-Glioma-associated oncogene homolog zinc finger protein (Hh-Gli)a signaling pathway, has attracted drug discovery scientists for the past decade. Hh-Gli signaling plays an important role in the embryonic patterning and development of many tissues and somatic structures as well as maintaining and repairing mature tissues in adults.2-4 Uncontrolled activation of the Hh-Gli pathway has been implicated in several cancers, including medulloblastoma, rhabdomyosarcoma, melanoma, basal cell carcinoma, and breast, lung, liver, stomach, prostate, and pancreatic cancers. 2, 5-8 Inhibition of the aberrant Hh-Gli pathway (Figure 1) has thus emerged as an attractive target for anticancer therapy.9-11 One Hh pathway inhibitor has shown promising results in phase I clinical trials and is proceeding to phase II12 studies, and two other compounds have entered phase I clinical trials.13, 14 In this article, we review the medicinal chemistry efforts to identify and design inhibitors of Hh-Gli signaling and present a perspective of future developments in this dynamic field. We also present a brief overview of the role of Hh-Gli signaling pathway in normal development and cancer. n n n nFigure 1 n nHedgehog pathway activators and inhibitors n n n nThe hedgehog (Hh) gene was first identified during a search for embryonic lethal mutants of Drosophila melanogaster, which found that mutation of Hh resulted in altered segment patterning of the larva.15 Subsequently the gene was identified in many other invertebrates and vertebrates, including humans. Three mammalian counterparts of the Hh gene, termed Sonic hedgehog (Shh), Dessert hedgehog (Dhh), and Indian hedgehog (Ihh), were identified by combined screening of mouse genomic and cDNA libraries.16 Hh undergoes multiple processing events, including autocatalytic cleavage of the C-terminal domain combined with addition of a cholesterol moiety at the cleavage site, and an N-terminal palmitoylation, to generate the active ligand.17-19 n nThe receptor of secreted Hh protein is the multipass transmembrane protein Patched (Ptch). Of the two vertebrate homologs of Ptch, Ptch1 and Ptch2, the role of Ptch1 is better understood. In the absence of Hh ligand, Ptch inhibits the activity of the downstream effector Smoothened (Smo). The binding of Hh inactivates Ptch, resulting in activation of Smo.20 In Drosophila, a complex of proteins comprising Fused (Fu), Suppressor of Fused (SuFu), and Costal-2 (Cos2) mediates signaling downstream of Smo and is aided by several kinases, such as protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1). Mammalian homologs of Fu and Cos2 have not yet been identified, suggesting that the signaling mechanisms differ in mammals and Drosophila. 21, 22 Several mammalian-specific kinases that are required for Shh signaling have been identified.23-25 These proteins modulate the function of Gli (Ci in Drosophila), the only transcription factor identified to date that operates directly downstream of Hh. n nThe first vertebrate Gli gene to be discovered was human Gli1, which was amplified about 50-fold in a malignant glioma.26 Vertebrates have three Gli proteins (Gli1, Gli2 and Gli3), all of which have five highly conserved tandem zinc fingers, a fairly conserved N-terminal domain, several potential PKA sites, and a number of additional small conserved regions in the C-terminal end. Despite these similarities, the functions of the Gli subtypes differ. Both Gli2 and Gli3 contain activation and repressor domains. Consequently, in the absence of upstream Hh signal, full-length Gli3 and, to a lesser extent, Gli2, are constitutively cleaved to generate a truncated repressor form.27-29 Hh signaling inhibits this cleavage, resulting in full-length Gli2 and Gli3, which have activator function. Gli1, in contrast, does not undergo proteolytic cleavage and acts as a constitutive activator.27 The transcription of Gli1 gene is initiated by Hh and is also controlled by Gli3.27 Target genes of the Hh pathway other than Gli1 include Ptch, several Wnt and TGFβ superfamily proteins, cell cycle proteins such as cyclin D, and stem-cell marker genes such as NANOG and SOX2.30, 31 Investigators are now attempting to comprehensively identify the Gli1-target genes.32, 33

Structure-activity relationships and cancer-cell selective toxicity of novel inhibitors of glioma-associated oncogene homologue 1 (Gli1) mediated transcription.

We report novel inhibitors of Gli1-mediated transcription as potential anticancer agents. Focused chemical libraries were designed and assessed for inhibition of functional cell-based Gli1-mediated transcription and selective toxicity toward cancer cells. The SAR was revealed, and the selectivity of the lead compounds inhibition of Gli1-mediated transcription over that of Gli2 was determined. Compound 63 (NMDA298-1), which inhibited Gli1-mediated transcription in C3H10T1/2 cells with an IC(50) of 6.9 muM, showed 3-fold selectivity for inhibiting transcription mediated by Gli1 over that by Gli2. Cell-viability assays were performed to evaluate the chemical library in a normal cell line and a panel of cancer cell lines with or without up-regulated expression of the Gli1 gene. These compounds decreased the viability of several cancer cell lines but were less active in the noncancerous BJ-hTERT cells.

Potential Strategies to Target Protein–Protein Interactions in the DNA Damage Response and Repair Pathways

This review article discusses some insights about generating novel mechanistic inhibitors of the DNA damage response and repair (DDR) pathways by focusing on protein-protein interactions (PPIs) of the key DDR components. General requirements for PPI strategies, such as selecting the target PPI site on the basis of its functionality, are discussed first. Next, on the basis of functional rationale and biochemical feasibility to identify a PPI inhibitor, 26 PPIs in DDR pathways (BER, MMR, NER, NHEJ, HR, TLS, and ICL repair) are specifically discussed for inhibitor discovery to benefit cancer therapies using a DNA-damaging agent.