Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140.
Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC50 values between 1.0 and 6 μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9-Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, United States. Electronic address: rafiqahaq@gmail.com.
Rafiqa
R
Eachkoti
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, United States.
M V Ramana
MV
Reddy
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, United States.
Yen K
YK
Lieu
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, United States.
Stephen C
SC
Cosenza
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, United States.
E PremKumar
EP
Reddy
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D047188:Chalcones / Q000494:pharmacology*; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D049409:Luciferases, Firefly / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction; D013450:Sulfones / Q000494:pharmacology*
D047188:Chalcones; D018841:HSP90 Heat-Shock Proteins; C000592163:ONO4140; D013450:Sulfones; D049409:Luciferases, Firefly
Firefly luciferase; Heat shock factor 1; Hsp90 inhibitors; Rabbit reticulolysate; Renaturation