Potency and Selectivity Assessment of Small Molecules Against Janus Kinase (JAK) 2: Widely Used AG490 Inhibitor Is Neither Potent Nor Selective for JAK2

Abstract 4780

Janus kinases (JAK) are the most proximal signaling components of multiple cytokine receptors and have four family members JAK1, JAK2, JAK3 and TYK2. JAK2 is essential for the development of normal erythroid and myeloid lineages by mediating signaling though the erythropoietin receptor (EPOR), thrombopoietin receptor (TPOR) and the β-common chain of GM-CSF, IL-3 and IL-5. Recently, JAK2 has been the focus of considerable research due to the discovery that patients with myeloproliferative disorders (MPDs) essential thrombocythemia, polycythemia vera and myelofibrosis contain somatically derived inactivating mutations in the JAK2 pseudokinase repressor domain. The deregulated expansion of erythro/myeloid cells in MPDs is thought to be due to the sustained signaling though JAK2 and downstream STAT, PI3K and MAPK signaling pathways to enhance the proliferation, survival and differentiation of progenitor cells. As a consequence, the discovery and development of small molecule inhibitors for JAK2 has been a focus for potential therapeutic intervention and has provided tools to examine cytokine networks.

In order to discover small molecule JAK2 inhibitors we evaluated a number of benchmark and commercially available inhibitors as well as new inhibitors we generated. Tyrphostin AG490 has been widely used in the literature as a “JAK2” inhibitor in EPOR signaling and MPD research. However, AG490 has also been reported as an inhibitor of JAK3, EGFR, HER2, guanylyl cyclase C and BCR-ABL. In JAK2 enzyme assays, our new JAK inhibitors AMG-Jak2-02 and AMG-Jak2-03 were found to have transit IC₅₀ <0.005 μM. However, AG490 was considered inactive in JAK2 enzyme assays with an IC₅₀ >125 μM. When profiled against other JAK family members in enzyme assays, AG490 was also considered inactive on JAK1 (IC₅₀ >125 μM), JAK3 (IC₅₀ >80 μM) and TYK2 (IC₅₀ >80 μM) whereas the IC₅₀ of AMG-Jak2-02 and AMG-Jak2-03 were between 0.02 – 1.1 μM in JAK enzyme assays. Due to the lack of inhibitory activity of AG490 in JAK family enzyme assays, we performed a broader kinase screen on AG490. AG490 was profiled at 1 μM against 48 kinases. To our surprise, it was inactive on all kinases tested (most potent was SGK1 with ∼20% inhibition). In a broader binding screen of 441 lipid and protein kinases, 25 μM of AG490 was considered active on 4 kinases: STK17A, STK17B, PDGFRA and PDGFRB with a >70% inhibition. AG490 was inactive on all JAK family members. To investigate the potential for AG490 to inhibit JAK2 in a cellular context we examined the phosphorylation (p) of downstream molecules STAT5, AKT and ERK1/2 in an EPO dependent cell line UT-7/EPO. UT-7/EPO cells were incubated with AG490 (dose range up to 100 μM) or other JAK inhibitors (doses up to 33 or 100 μM) and phosphorylation of downstream molecules was assessed using Western immunoblot analysis. The EPO-EPOR induced downstream generation of pSTAT5, pAKT and pERK1/2 was suppressed by pan-JAK inhibitor I (Calbiochem) with an IC₅₀ ∼ 0.1 μM, by AMG-Jak2-02 with an IC50 ∼ 10 μM and by AMG-Jak2-03 with an IC₅₀ ∼ 0.1 μM. However, AG490 at 100 μM was unable to suppress the EPO-EPOR induced generation of pERK1/2 or pAKT but had modest effects on suppressing the generation of pSTAT5 (IC₅₀ between 50–100 μM). We also investigated the potential for AG490 to inhibit the viability of JAK2 dependent (UT-7/EPO) and JAK2-independent (γ2A JAK2 null) cells. Pan JAK inhibitor I (Calbiochem), AMG-Jak2-01 and AMG-Jak2-02 were > 10 fold more potent at reducing JAK2 dependent cell viability (UT-7/EPO cells) compared with the viability of JAK2 independent cell line (γ2A cells). However, AG490 was found to be equipotent at inhibiting the viability of JAK2 dependent and independent cell lines. Similar results were obtained when these studies were repeated multiple times using multiple lots of compound (confirmed to be structurally correct based on NMR analysis).

Taken together, we have identified that the widely used “JAK2” inhibitor AG490 is neither potent nor selective for JAK2. Thus, published data generated with AG490 should be interpreted with caution. Careful validation of JAK2 compounds for future research and assay development purposes should be taken into consideration.