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Drug combination testing in the pharmaceutical industry has typically been driven by late-stage opportunistic strategies rather than by early testing to identify drug combinations for clinical investigation that may deliver improved efficacy. A rationale for combinations exists across a number of diseases in which pathway redundancy or resistance to therapeutics are evident. However, early assays are complicated by the absence of both assay formats representative of disease biology and robust infrastructure to screen drug combinations in a medium-throughput capacity. When applying drug combination testing studies, it may be difficult to translate a study design into the required well contents for assay plates because of the number of compounds and concentrations involved. Dispensing these plates increases in difficulty as the number of compounds and concentration points increase and compounds are subsequently rolled onto additional labware. We describe the development of a software tool, in conjunction with the use of acoustic droplet technology, as part of a compound management platform, which allows the design of an assay incorporating combinations of compounds. These enhancements to infrastructure facilitate the design and ordering of assay-ready compound combination plates and the processing of combinations data from high-content organotypic assays.

The concept of harnessing the immune system to target cancer cells has been an active area of research for decades. The advent of antibodies targeting the checkpoint receptors CTLA-4 and PD-1/PDL-1, have now provided definitive clinical validation for this approach. Since these discoveries the search for small molecule immuno-oncology agents have intensified. Here we present data on HPK1 (hematopoietic progenitor kinase 1), a novel immuno-oncology kinase involved in the negative regulation of T-cell receptor (TCR) signalling and describe a high throughput screen to identify novel chemical starting points to develop potent and selective inhibitors of this kinase. HPK1 is a member of the Ste-20 family of Serine/Threonine protein kinases. It is expressed highly in cells of haematopoietic lineage, including T-cells and is activated upon engagement of the TCR with cell surface MHC complexes. Active HPK1 leads to phosphorylation of an adaptor protein SLP76, triggering a signalling cascade that results in the downregulation of TCR signalling and thus downregulation of T-cell function. Recent reports have demonstrated that the kinase activity is essential for the function of HPK1 in T-cells and support the hypothesis that small molecule inhibitors of HPK1 kinase activity will result in sustained activation of T-cells. To identify inhibitors of HPK1 we developed an enzyme activity assay that could be used in a high throughput assay to screen Sygnature Discovery's proprietary screening library. We utilised the intrinsic ATPase activity of HPK1 to develop an ADP product fluorescent polarisation assay suitable for an HTS screen. We validated the assay using a series of known inhibitors with a broad range of potencies versus HPK1 activity. Next, we developed a cell-based assay encompassing anti-CD3 TCR mediated induction of phosphorylation of SLP76 in Jurkat cells. We tested the activity of our panel of HPK1 tool inhibitors in this assay and demonstrated a strong correlation between the biochemical and cell potencies of the compounds, further validating the robustness and cell potency predictivity of the biochemical assay. The biochemical assay was then used to screen our small molecule chemical library to identify potential inhibitors of HPK1 kinase activity. This library is biased toward lead-like chemical space and has undergone significant triage to remove unwanted chemotypes and PAINs moieties. The initial hits were confirmed and IC50 values determined for a select group of chemotypes. These compounds were subsequently tested in the Jurkat pSLP76 cell assay for cellular activity versus HPK1. In summary we present the results of a successful HTS and hit evaluation to identify novel and attractive chemical starting points for the development of inhibitors of the immuno-oncology target HPK1. Citation Format: Denise Swift, Samantha Hitchin, Chris Tomlinson, Anindita Sengupta, Callum Taylor, Alec O'Keeffe, Grace Boden, John Unitt, Stuart Thomson, Allan M. Jordan. A high throughput screen of the novel immuno-oncology target HPK1 identifies a range of chemical starting points for the development of potent and selective inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5317.