The HPK1 Inhibitor A-745 Verifies the Potential of Modulating T Cell Kinase Signaling for Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.

Small Molecule and Pooled CRISPR Screens Investigating IL17 Signaling Identify BRD2 as a Novel Contributor to Keratinocyte Inflammatory Responses.

Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of ∼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.

Identification and characterization of mGlu3 ligands using a high throughput FLIPR assay for detection of agonists, antagonists, and allosteric modulators.

When targeting G-protein coupled receptors (GPCRs) in early stage drug discovery, or for novel targets, the type of ligand most likely to produce the desired therapeutic effect may be unknown. Therefore, it can be desirable to identify potential lead compounds from multiple categories: agonists, antagonists, and allosteric modulators. In this study, we developed a triple addition calcium flux assay using FLIPR Tetra to identify multiple ligand classes for the metabotropic glutamate receptor 3 (mGlu3), using a cell line stably co-expressing the human G-protein-coupled mGlu3 receptor, a promiscuous G-protein (G(α16)), and rat Glast, a glutamate transporter. Compounds were added to the cells followed by stimulation with EC(10) and then EC(80) concentration of glutamate, the physiological agonist for mGlu receptors. This format produced a robust assay, facilitating the identification of agonists, positive allosteric modulators and antagonists/negative allosteric modulators. Follow up experiments were conducted to exclude false positives. Using this approach, we screened a library of approximately 800,000 compounds using FLIPR Tetra and identified viable leads for all three ligand classes. Further characterization revealed the selectivity of individual ligands.

A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen.

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.