A Case Study from the Chemistry Core of the Pittsburgh Molecular Library Screening Center: The Polo-like Kinase Polo-Box Domain (Plk1-PBD)

The Polo-like kinase (Plk) family comprises four cell cycle serine/threonine kinases, Plk1-4. Among these, Plk1 has been most thoroughly characterized; it contains a conserved kinase domain and a C-terminal docking site for S/T-phosphorylated proteins (polo-box domain, PBD). Polo-like kinases are deregulated in oncogenesis and therefore constitute a therapeutic target for cancer. A high throughput screening campaign was carried out by the Pittsburgh Molecular Library Screening Center (PMLSC), using a fluorescence polarization assay with recombinant Plk1-PBD to monitor the inhibition of binding of an optimal phosphopeptide substrate motif with recombinant Plk1-PBD. Screening of 97,090 small molecule library samples provided by the NIH Small Molecule Repository distributed by DPI Galapagos led to 11 confirmed hits. The Pittsburgh MLSCN Chemistry Core selected one of the structurally most tractable hits, SID 861574, for chemical hit-to-probe development. A broad chemistry program was initiated that developed new strategies for 6-amino- and 6-hydroxy uracil synthesis as well as acylanilides, and generated a total of 70 analogs. Out of 46 analogues tested, none, nor the resynthesized hit, showed affinity to Plk1-PBD in the follow up assays. In contrast, re-assays of the original screening materials displayed activities similar to the original HTS assay. We ultimately concluded that an impurity in the commercial material led to the positive screening artifact. This case study highlights our development of a synthesis of 6-position functionalized uracil analogs, but also illustrates the importance of careful quality and compound stability monitoring of screening collections.