Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

Abstract

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. 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. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time-and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.

The Stilbenoid Tyrosine Kinase Inhibitor, G6, Suppresses Jak2-V617F-mediated Human Pathological Cell Growth in Vitro and in Vivo

“…Jak2/STAT signaling is known to positively regulate cell growth by directly increasing expression of the anti-apoptotic marker, Bcl-xL, via STAT-binding elements located in its proximal promoter region (26,27). In a recently published work, we reported that G6 inhibits HEL cell growth via the down-regulation of Bcl-xL and this correlates with significantly reduced phospho-STAT5 levels (28). Furthermore, we showed that G6 treatment of HEL cells results in upregulation of pro-apoptotic Bim, and cleavage of pro-apoptotic Bid, from its inactive precursor to its active form (28).…”

“…In a recently published work, we reported that G6 inhibits HEL cell growth via the down-regulation of Bcl-xL and this correlates with significantly reduced phospho-STAT5 levels (28). Furthermore, we showed that G6 treatment of HEL cells results in upregulation of pro-apoptotic Bim, and cleavage of pro-apoptotic Bid, from its inactive precursor to its active form (28). In our work here, we show that G6 treatment results in reduced STAT5 phosphorylation within HEL cells (Fig.…”

Identification of novel SAR properties of the Jak2 small molecule inhibitor G6: Significance of the para-hydroxyl orientation

“…16 We also demonstrated that the stilbene core structure of G6 is essential for its activity as a Jak2 inhibitor, in vitro and ex vivo . 17 Here, we further examined the structure of G6 to determine if structural modifications of the compound could enhance its Jak2 inhibitory potential. We hypothesized that the structure of G6 could be minimized in order to enhance its Jak2 inhibitory potential and that maintaining the para -hydroxyl orientation is required for Jak2 inhibition.…”

“…NB13 and NB15 showed interactions very similar to those of the parent compound, G6. 17 These include hydrogen bonds with residues in the hinge region, the activation loop, and the catalytic loop. NB4 and NB6 showed hydrogen bond interactions with several residues, but lacked any interactions with the critical hinge region residues, Glu930 or Leu932.…”

“…21–22 We have previously shown that G6 reduces STAT5 phosphorylation in HEL cells. 17 Therefore, we wanted to determine whether specific structural features in G6 correlate with reduced STAT5 phosphorylation in these cells. Fig.…”