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Application: Study enzyme kinetics and screen small molecule inhibitors for drug discovery and high throughput screening (HTS) applications.
Background: HPK1 (Hematopoietic progenitor kinase 1), also known as MAP4K1, is a restricted member of the Ste20 serine/threonine kinase super family expressed in the hematopoietic cell lineage, with high expression in T, B and dendritic cells (DCs). It is considered a negative intracellular immune checkpoint as HPK1 diminishes T cell receptor (TCR) signaling activity and T cell proliferation. Cytosolic HPK1 is recruited to the plasma membrane when the TCR is activated, is phosphorylated, then phosphorylates the adaptor protein SLP-76 (SH2 domain containing leukocyte protein of 76 kDa), forming a site for the negative regulator 14-3-3 to bind and destabilize the TCR complex. It also plays a scaffolding role in NF-κB (nuclear factor kappa light-chain-enhancer of activated B cells) regulation in AICD (activation-induced cell death) and LFAT-1 (lymphocyte function-associated antigen 1) activation. Knock-down or knock-out of HKP1 in animals resulted in improved anti-viral and anti-tumor immunity, suggesting that HPK1 could be a novel target for anti-tumor immunotherapy. The development of small molecule inhibitors specific for HPK1 has not been easy due to its similarity to other MAP4K proteins. Additionally, typical off-target management links to the minimization of effects on crucial functions for life, while in the case of HPK1 one must consider effects on functions that support cancer cell killing. The use of allosteric inhibitors, CRISPR, shRNA, ASO (antisense oligonucleotides) or PROTAC (Proteolytic targeting chimeras) are also interesting alternatives that can be used to target HPK1. Recently, the results of a phase I/II clinical trial for NDI-101150, a selective inhibitor of HPK1, have indicated an increased infiltration of CD8+ T cells and DCs into RCC (renal cell carcinoma), showcasing the role and importance of HPK1 in oncology.
Contraindications: The final concentration of DMSO in the assay should not exceed 1%.
Description: The Chemi-Verse™ HPK1 Kinase Assay Kit is designed to measure HPK1 (Hematopoietic progenitor kinase 1) serine/threonine kinase activity for screening and profiling applications using ADP-Glo™ as a detection reagent. The assay kit comes in a convenient 96-well format, with enough purified HPK1 (amino acids 1-346, kinase domain), kinase substrate, ATP, and kinase assay buffer for 100 enzyme reactions.Note: MBP (Myelin Basic Protein) is a non-specific protein substrate that is used as a "universal substrate" for many in vitro kinase activity assays. This protein is targeted by many serine/threonine kinases at conserved amino acids. We use the dephosphorylated version of the MBP substrate in our assays to determine the kinase-mediated phosphorylation of MBP. Our assays are not suitable for studying autophosphorylation of the kinase due to the presence of the MBP substrate.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Uniprot: Q92918
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
References: Sawasdikosol S. and Burakoff S., 2020 eLife: 55122.
You D., et al., 2021 J Immunother Cancer 9(1):e001402.
Noel M., et al., 2024 Journal of Clinical Oncology 42(16):suppl
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