ABSTRACT
The discovery and development of a series of thiophenes as potent and selective inhibitors of PLK is described. Identification and characterization of 2, a useful in vitro PLK inhibitor tool compound, is also presented.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiophenes/antagonists & inhibitors , Animals , Cell Cycle , Cell Cycle Proteins/chemistry , Cell Line, Tumor , Cell Proliferation , Drug Design , Humans , Inhibitory Concentration 50 , Mice , Mitosis , Models, Chemical , Molecular Conformation , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Thiophenes/chemistry , Polo-Like Kinase 1ABSTRACT
A series of thiophene PLK1 inhibitors was optimized for increased solubility and reduced protein binding through the appendage of basic amine functionality. Interesting selectivity between PLK1 and PLK3 was also obtained through these modifications.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiophenes/chemistry , Cell Cycle , Cell Cycle Proteins/chemistry , Cell Line, Tumor , Drug Design , Humans , Inhibitory Concentration 50 , Mitosis , Models, Chemical , Molecular Conformation , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Solubility , Tumor Suppressor Proteins , Polo-Like Kinase 1ABSTRACT
Polo-like kinase 1 (PLK1) plays key roles in the regulation of mitotic progression, including mitotic entry, spindle formation, chromosome segregation, and cytokinesis. PLK1 expression and activity are strongly linked to proliferating cells. Many studies have shown that PLK1 expression is elevated in a variety of tumors, and high expression often correlates with poor prognosis. Using a variety of methods, including small-molecule inhibition of PLK1 function and/or activity, apoptosis in cancer cell lines, cell cycle arrest in normal cell lines, and antitumor activity in vivo have been observed. In the present study, we have examined the in vitro biological activity of a novel and selective thiophene benzimidazole ATP-competitive inhibitor of PLK1 and PLK3 (5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benzyl]oxy}thiophene-2-carboxamide, called compound 1). Compound 1 has low nanomolar activity against the PLK1 and PLK3 enzymes and potently inhibits the proliferation of a wide variety of tumor cell lines. In the lung adenocarcinoma cell line NCI-H460, compound 1 induces a transient G(2)-M arrest, mitotic spindle defects, and a multinucleate phenotype resulting in apoptosis, whereas normal human diploid fibroblasts arrest in G(2)-M and show little apoptosis. We also describe a cellular mechanistic assay that was developed to identify potent intracellular inhibitors of PLK1. In addition to its potential as a therapeutic agent for treating cancer, compound 1 is also a useful tool molecule for further investigation of the biological functions of PLK1 and PLK3.
Subject(s)
Adenocarcinoma/drug therapy , Benzimidazoles/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Lung Neoplasms/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiophenes/pharmacology , Adenocarcinoma/enzymology , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/pharmacology , Apoptosis/drug effects , Benzimidazoles/chemistry , Binding, Competitive , Blotting, Western , Cell Cycle/drug effects , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescent Antibody Technique , Humans , Immunoblotting , Lung Neoplasms/enzymology , Microscopy, Fluorescence , Molecular Structure , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Thiophenes/chemistry , Tumor Suppressor Proteins , Polo-Like Kinase 1ABSTRACT
Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.
Subject(s)
Antineoplastic Agents/pharmacology , Oxadiazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Female , Humans , Mice , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/metabolism , Oxadiazoles/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.
Subject(s)
Oxadiazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Animals , Female , Magnetic Resonance Spectroscopy , Mass Spectrometry , Mice , Mice, SCID , Models, Molecular , Oxadiazoles/chemistry , Oxadiazoles/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Substrate SpecificityABSTRACT
The shift in apparent IC(50) that attends addition of serum proteins to in vitro cellular, enzymatic, and receptor binding assays can be used to determine the dissociation constant for compound-serum protein complexes. We show here that a simple linear relationship exists between the apparent IC(50) in the presence of serum protein and the inverse of the apparent K(d) for the compound-serum protein complex. Using a series of cell-active kinase inhibitors we demonstrate that the K(d) value derived in this way can be used to predict the extent of protein binding in serum for various compounds. This method should provide a simple means of assessing the relative serum protein binding propensity of compounds early in the compound optimization phase of drug discovery campaigns.