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

Abstract

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.

The Essential Roles of Chemistry in High-Throughput Screening Triage

“…Another related issue is the hydration of compounds stored in dimethyl sulfoxide (DMSO) and the effect of freeze-thaw cycles [142][143][144]. We strongly recommend re-checking vendor-supplied samples for purity (e.g., HPLC-MS) and structural characterization ( 1 H and 13 C NMR) [145,146]. Ideally, this would be done for every commercial re-supply, but in practice this is difficult for smaller academic labs, and therefore we recommend this be a priority for those compounds that have high probabilities of being recommended for mechanism-of-action studies or analog expansion.…”

“…However, compound samples with even trace impurities can lead project teams astray. These impurities can be remnants from the chemical synthesis, including transition metal catalysts, leftover starting materials, reactive intermediates, oxidative products, rearrangements or enantiomers, to name a few [146][147][148][149][150][151][152][153]. Therefore, medicinal chemists should insist on in-house HPLC purification (or comparable method) of any compound making its way into critical experiments.…”

Addressing Compound Reactivity and Aggregation Assay Interferences: Case Studies of Biochemical High-Throughput Screening Campaigns Benefiting from the National Institutes of Health Assay Guidance Manual Guidelines

“…Furthermore, compound stock solutions can contain impurities, including degradation products, metals, and remnants of reactive reagents used in the compound synthesis that interfere with assays or cause nonspecific activity. 8–11 Compound interferences, including nonspecific reactivity and aggregation, can adversely affect both biochemical (cell-free) and cellular (including phenotypic and high-content assays) assays. 12…”

Post-HTS case report and structural alert: Promiscuous 4-aroyl-1,5-disubstituted-3-hydroxy-2 H -pyrrol-2-one actives verified by ALARM NMR

“…1 However, despite their potential utility, rarely are the details of the decision-making process behind ‘go/no-go’ decisions described in the scientific literature. 2–4 …”