Daniel F. Hassler

In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1

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 G2-M arrest, mitotic spindle defects, and a multinucleate phenotype resulting in apoptosis, whereas normal human diploid fibroblasts arrest in G2-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. [Mol Cancer Ther 2007;6(2):450–9]

Design of potent thiophene inhibitors of polo-like kinase 1 with improved solubility and reduced protein binding.

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.

Discovery of thiophene inhibitors of polo-like kinase.

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.

Imidazo[5,1-f][1,2,4]triazin-2-amines as novel inhibitors of polo-like kinase 1

The synthesis and biological activities of imidazo[5,1-f][1,2,4]triazin-2-amines (imidazotriazines) as novel polo-like kinase 1 inhibitors are reported.

A high throughput sphingomyelinase assay.

Publisher Summary Sphingomyelinases (SMases) are phophodiesterases that catalyze hydrolysis of the phosphodiester linkage of sphingomyelin (SM) to yield ceramide or ceramide-l-phosphate as the lipidic product and phosphorylcholine or choline as the headgroup product. SMases have been assayed using a variety of substrates. Radiolabeled sphingomyelin, prepared via incorporation of 3 H or 14 C into the headgroup moiety, is the most common substrate. Colorimetric and fluorescent assays have been developed that utilize SM analogs containing chromophores or fluors. However, these classes of substrates differ significantly from authentic SM; these are not widely used. Assays employing radiolabeled substrate require physical separation of product from substrate. Usually this separation is accomplished with liquid-liquid phase extractions that effectively separate lipophilic substrate and hydrophilic product into organic and aqueous phases, respectively. The reaction components can also be separated chromatographically. These methods are tedious and are not suitable for large numbers of samples. An alternate method employs headgroup radiolabeled SM with separation of the unreacted substrate from the labeled product by precipitation. In this strategy, the labeled substrate is selectively precipitated while the labeled headgroup product remains soluble. The latter can then be transferred easily for quantitation by liquid scintillation counting. The advantage of this method is the ease with which multiple samples can be processed simultaneously. This chapter describes methods of exploiting this separation technique to greatly increase the efficiency and throughput of SMase assays.