Supplementary Tables 1-3 from Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

Additions to Supplement Table 3 from Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

Abstract Polo-like kinase 1 (Plk1) is a conserved serine/threonine kinase that plays an essential role in regulating the many processes involved in mitotic entry and progression. In humans, Plk1 is expressed primarily during late G2 and M phases and, in conjunction with Cdk1/cyclin B1, acts as master regulatory kinases for the myriad protein substrates involved in mitosis. Plk1 overexpression is strongly associated with cancer and has been correlated with poor prognosis in a broad range of human tumor types. We have identified a potent, selective, reversible, ATP-competitive inhibitor of Plk1, GSK461364A, capable of inhibiting cell growth of most proliferating cancer cell lines tested. We observe distinct cell cycle effects of GSK461364A depending on the dose used. The predominant phenotype for cells treated with GSK461364A is prometaphase arrest with characteristic collapsed polar polo spindle. At high concentrations, GSK461364A delays mitotic entry in G2 followed by gradual progression into terminal mitosis; in some cell lines, this correlates with decreased apoptosis. Cell culture growth inhibition by GSK461364A can be cytostatic or cytotoxic but leads to tumor regression in xenograft tumor models under proper dose scheduling. Finally, we describe pharmacodynamic biomarkers of GSK461364A activity (pHH3 and Plk1) that are currently being evaluated in human cancer clinical trials. [Cancer Res 2009;69(17):6969–77]

3257 Polo-like kinases are evolutionarily conserved ser/thr kinases that play critical roles in regulating mitosis in diverse organisms from yeast to mammalian cells. In human cells, Polo-like kinase-1 (Plk1) has been implicated in a variety of different processes, including mitotic entry, spindle formation and cytokinesis. Overexpression of Plk1 is strongly associated with cancer and correlates with poor prognosis in some tumor types, and it is therefore a target of chemotherapeutic intervention. We have identified GSK461364 as a novel thiophene amide inhibitor of Plk1 which has excellent potency against a variety of tumor cell lines, is efficacious in PD models, and shows in vivo tumor inhibition in animal models. In order to interrogate the affinity and kinetics of this inhibitor, we developed and characterized a catalytic activity assay for full length Plk1 by formation of a complex with a polobox domain binding phospho-peptide. Under these assay conditions, GSK461364 was found to be a potent, ATP-competitive inhibitor of full length Plk1 enzyme with a Ki of 2.2 ± 0.6 nM. When evaluated against full-length Plk2 and Plk3, the inhibitor exhibited approximately 400-fold greater potency for Plk1. When tested against a panel of 48 additional kinases, it was at least 100-fold selective for Plk1. GSK461364 was also found to form a rapidly reversible complex with Plk1. Our data suggest that the excellent cellular activity and in vivo efficacy demonstrated by this compound is a result of the potent and selective inhibition of Plk1, and support the advancement of GSK461364 as a novel Plk1 inhibitor for the treatment of cancer.

ABSTRACTRetapamulin is a semisynthetic pleuromutilin derivative being developed as a topical antibiotic for treating bacterial infections of the skin. It is potent in vitro against susceptible and multidrug-resistant organisms commonly associated with bacterial skin infections. We report detailed mode of action studies demonstrating that retapamulin binds to the bacterial ribosome with high affinity, inhibits ribosomal peptidyl transferase activity, and partially inhibits the binding of the initiator tRNA substrate to the ribosomal P-site. Taken together, these data distinguish the mode of action of retapamulin from that of other classes of antibiotics. This unique mode of action may explain the lack of clinically relevant, target-specific cross-resistance of retapamulin with antibacterials in current use.