Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.
Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/β)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90β/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/β)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.
Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA 94305, USA.
Carmel T
CT
Chan
Robert E
RE
Reeves
Ron
R
Geller
Shahriar S
SS
Yaghoubi
Aileen
A
Hoehne
David E
DE
Solow-Cordero
Gabriela
G
Chiosis
Tarik F
TF
Massoud
Ramasamy
R
Paulmurugan
Sanjiv S
SS
Gambhir
D000081:Acetamides / Q000494:pharmacology*; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D055808:Drug Discovery; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D007093:Imidazoles; D047468:Immunoprecipitation; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D007854:Lead / Q000494:pharmacology; D049409:Luciferases, Firefly; D049410:Luciferases, Renilla; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D049268:Positron-Emission Tomography; D000071876:Prostaglandin-E Synthases; D017510:Protein Folding; D020033:Protein Isoforms / Q000378:metabolism; D011719:Pyrazines; D054852:Small Molecule Libraries; D013853:Thioacetamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology*; D014057:Tomography, X-Ray Computed; D014316:Tritium
D000081:Acetamides; D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D007093:Imidazoles; D047029:Lactams, Macrocyclic; C576667:N-(5-methylisoxazol-3-yl)-2-(4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio)acetamide; D020033:Protein Isoforms; D011719:Pyrazines; D054852:Small Molecule Libraries; D013876:Thiophenes; D013853:Thioacetamide; D014316:Tritium; D007854:Lead; C017144:coelenterazine; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; C001277:geldanamycin