Finally, compound 12k and 12l were identified as the promising inhibitors of JAK2, which exhibited high inhibitory activity (IC<sub>50</sub> = 6 nM and 3 nM, respectively) and selectivity for JAK2 over JAK1 and JAK3, and showed potent antiproliferative activities toward HEL human erythroleukemia cells.
We investigated the activity of (E)-3(6-bromopyridin-2-yl)-2-cyano-N-(S0-1phenylethyl)acrylamide (WP1066), a novel analogue of the JAK2 inhibitor AG490, in JAK2V617F-positive erythroleukemia HEL cells and in blood cells from patients with polycythemia vera.
The leukemic CD71<sup>+</sup> cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2V617F-transduced p53-null erythroleukemia.
These results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.
Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo.
Using this assay and serial dilutions of an erythroleukemia cell line harboring the homozygous JAK2V617F mutation, we successfully detected the mutation within a background of wild type sequences at a sensitivity of 2.5%.