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Fujii Lab: Research

Functional small molecule antagonist of Dishevelled

Dvl protein is surprisingly multi-functional central hub of Wnt signaling pathways (interacting with >33 proteins). Overexpression of Dvl alone is sufficient to activate Tcf transcription, the canonical Wnt pathway. Oncogenic enhancement of Tcf transcription, such as constitutive activation of beta-catenin and overexpression of Dvl, is observed in many cancers. Tcf transcriptional activation has been implicated also in diabetes (potently inhibits adipogenesis) and hepatic fibrosis (activates hepatic stellate cells). The PDZ domain is the Dvl domain most often found to interact with Dvl-binding proteins, and it controls both the canonical and non-canonical Wnt pathways. We have designed the first non-peptide small molecule antagonist of the Dvl PDZ domain interaction, FJ9, which downregulates the Dvl-activated Tcf transcription, causes apoptosis beta-catenin dependently, and suppresses tumor growth in mice xenograft models. This is a still rare example of protein-protein interaction antagonist that can perform a therapeutic response in animal. We also have produced chemical libraries by utilizing existing hypotheses about binding mode and structure activity relationships, and generated second lead compound with improved in vivo efficacy.

Functional small molecule antagonist of Dishevelled

Small molecule-based chemical biology for disease-related PDZ domains

The PDZ domain is a common protein-protein interaction module utilized in diverse cell signaling pathways. Human genome is believed as encoding >1000 PDZ domains, in which many of them are responsible in diverse human diseases. Three collaborative programs are ongoing to discover small molecule compounds that selectively target such PDZ domain containing proteins, including NHERF, syntenin, PDZK1, and MRT1. One of those compounds significantly suppressed oncogenic transformation of mammalian cells by a viral oncoprotein of human T-cell lymphotropic virus, a same class of retrovirus as the AIDS virus. We also have designed chemical probes that selectively and covalently bind to PDZ domains. We will use those probes to determine the change of PDZ protein expression in tumor tissues to identify PDZ domain proteins that enhances the efficacy of radiation therapy.

PDZ Domain Probe

Small molecular therapeutic agents targeting Gli1 transcription

The Gli1 is a member of the family of the Gli-Kruppel transcription factors in the Sonic Hedgehog (Shh) pathway. Overexpression of the Gli1gene is frequently found in medulloblastoma patients, especially those younger than 3 years, who suffered very serious brain damage after receiving traditional radiation therapy (lowering of IQ, lifetime physical handicap). However, Gli1 knock-out mice do not have any pathological defects, suggesting that Gli1 is an oncoprotein specifically regulated in tumorigenesis. Several efforts have been made for inhibiting Shh pathway by using antagonist of Smoothened (Smo). A study has suggested that pediatric patients will not tolerate to Smo antagonist, as Smo activity is essential also for somatic development that was demonstrated in young mice whose bone growth has severely damaged by the Smo antagonist. We have been engaging a research program to find new small molecules that selectively inhibit transcription that is activated by Gli1 overexpression, not Smo antagonists. A compound, NMDA167-3, is a drug-like small molecule inhibitor of the Gli1 transcription, and induce apoptosis in cancer cells but not in non-cancerous cells.

Small molecular therapeutic agents targeting Gli1 transcription

Nonpeptide small molecule inhibitors of PCNA protein-protein interaction

Over the past decades, numerous cancer drug discovery programs have targeted specific oncogenic pathways to create small molecule inhibitors. Often these agents are not therapeutically efficacious in diverse cancers, presumably because tumors can be supported by multiple mechanisms, e.g., not 'addictive' to the pathway targeted. Recent efforts targeting non-oncogenic mediators that ubiquitously support cancers have proven to be successful; such as HDAC, HSP90, Aurora, ubiquitin ligase, splisosome, proteasome, etc. Proliferating Cell Nuclear Antigen (PCNA) is one of such targets that has been unexplored. PCNA organizes numerous components of cell cycle machinery and DNA replication by interacting with their PCNA-binding motif called a PIP-box. We have found a non-peptide small molecule that inhibits interaction of PCNA protein and a PIP-box sequence peptide. Direct binding of this compound to PCNA was verified by surface plasmon resonance. The co-crystal structure of PCNA and this compound shows that it binds to the same PCNA cavity that PIP-box protein sequences bind, Medicinal chemistry efforts have identified clear SAR and created several new lead compounds. One of them, named T2AA, showed inhibitory activity at IC50= 1 microM, and promoted S arrest in HeLa cells. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to PCNA bi-molecularly (Figure); one to the PIP-box binding cavity and another to the surface adjacent to Lys164, which is monoubiquitinated upon DNA damage response. T2AA inhibited monoubiquitinated PCNA interactions and repair of interstrand DNA crosslink (ICL) in cells. Structure-based lead optimization and functional validations have shown that those PCNA inhibitors are useful for inhibiting the translesion DNA synthesis, thereby possible novel mechanistic chemosensitizers for cancer therapeutics.

PCNA inhibitors are useful for inhibiting the translesion DNA synthesis