Several members of the kinesin family of microtubule motor proteins play essential roles in mitotic spindle function and are potential targets for the discovery of novel antimitotic cancer therapies. KSP, also known as HsEg5, is a kinesin that plays an essential role in formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. We identified a potent inhibitor of KSP, CK0106023, which causes mitotic arrest and growth inhibition in several human tumor cell lines. Here we show that CK0106023 is an allosteric inhibitor of KSP motor domain ATPase with a K i of 12 nM. Among five kinesins tested, CK0106023 was specific for KSP. In tumor-bearing mice, CK0106023 exhibited antitumor activity comparable to or exceeding that of paclitaxel and caused the formation of monopolar mitotic figures identical to those produced in cultured cells. KSP was most abundant in proliferating human tissues and was absent from cultured postmitotic neurons. These findings are the first to demonstrate the feasibility of targeting mitotic kinesins for the treatment of cancer.

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Several small-compound library subsets (14,000 to 56,000) have been established to complement screening of a larger Genentech corporate library (~1,300,000). Two validation sets (~1% of the total library) containing compounds representative of the main library were chosen by selection of plates or individual compounds. Use of these subsets guided selection of assay configuration, validated assay reproducibility, and provided estimates of hit rates expected from our full library. A larger diversity subset representing the scaffold diversity of the full library (3.4% of the total) was designed for screening more challenging targets with limited reagent availability or low-throughput assays. Retrospective analysis of this subset showed hit rates similar to those of the main library while recovering a higher proportion of hit scaffolds. Finally, a property-restricted diversity set called the "in-between library" was established to identify ligand-efficient compounds of molecular size between those typically found in fragment and high-throughput screening libraries. It was screened at fivefold higher concentrations than the main library to facilitate identification of less potent yet ligand-efficient compounds. Taken together, this work underscores the value of generating multiple purpose-focused, diversity-based library subsets that are designed using computational approaches coupled with internal screening data analyses to accelerate the lead discovery process.

Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed.

Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 μL scaled-down process, we validated 164 SMs to improve yields by up to twofold. The titer increase mediated by the SMs varied for different antibodies. SM dose optimizations resulted in almost threefold higher titers. The top 2, structurally distinct SM hits, increased antibody titers more than twofold in a 1 mL production process. Averaged across three antibodies of different expression levels, the compounds enhanced transient productivity by ∼80%. Intriguingly, both compounds arrested cells in the G2/M cell cycle phase leading to a decrease in growth and nutrient consumption, while elevating titer, nuclear plasmid DNA (pDNA) copy numbers, and mRNA levels. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 3:1579-1588, 2017.