gw-7647 and Fanconi-Anemia

Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

Topics: Antineoplastic Agents; DNA Damage; DNA Helicases; DNA Repair; Drug Screening Assays, Antitumor; Fanconi Anemia; High-Throughput Screening Assays; Humans; Multiprotein Complexes; Protein Interaction Maps; RecQ Helicases

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

Topics: Algorithms; Antineoplastic Agents; Arabidopsis Proteins; Butyrates; Cell Line, Tumor; Cell Survival; Cisplatin; Colony-Forming Units Assay; DNA Damage; DNA, Neoplasm; Drug Resistance, Neoplasm; Electrophoresis, Polyacrylamide Gel; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; High-Throughput Screening Assays; Humans; Indicators and Reagents; Nuclear Proteins; Phenylurea Compounds; Pimozide; Proliferating Cell Nuclear Antigen; Recombinant Proteins; Recombination, Genetic; RNA, Small Interfering; Sister Chromatid Exchange; Ubiquitin-Specific Proteases; Ubiquitination

Ubiquitin-specific proteases (USPs) have in recent years emerged as a promising therapeutic target class. We identified selective small-molecule inhibitors against a deubiquitinase complex, the human USP1/UAF1, through quantitative high throughput screening (qHTS) of a collection of bioactive molecules. The top inhibitors, pimozide and GW7647, inhibited USP1/UAF1 noncompetitively with a K(i) of 0.5 and 0.7 μM, respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase, and cysteine proteases. The USP1/UAF1 inhibitors act synergistically with cisplatin in inhibiting cisplatin-resistant non-small cell lung cancer (NSCLC) cell proliferation. USP1/UAF1 represents a promising target for drug intervention because of its involvement in translesion synthesis and Fanconi anemia pathway important for normal DNA damage response. Our results support USP1/UAF1 as a potential therapeutic target and provide an example of targeting the USP/WD40 repeat protein complex for inhibitor discovery.

Topics: Antineoplastic Agents; Arabidopsis Proteins; Butyrates; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cisplatin; DNA Repair; Drug Resistance, Neoplasm; Endopeptidases; Fanconi Anemia; Humans; Kinetics; Nuclear Proteins; Phenylurea Compounds; Pimozide; Ubiquitin; Ubiquitin-Specific Proteases