JAK/HDAC bispecific inhibitor developed to be efficient against solid tumors and malignant leukemia

The authors et al. investigated, designed, and synthesized 27 new JAK/HDAC dual-target inhibitors based on 2-amino-4-phenylaminopyrimidine (compounds 7–33), which are anticipated to increase the efficacy of single-target medications, take advantage of the synergistic effects of dual-target inhibitors in hematologic malignancies, and broaden the therapeutic range of dual-target inhibitors in solid tumors.

In vitro inhibitory activities against JAK2 and HDAC were examined using a series of compounds developed and optimized by the authors et al. The results showed that compounds 20, 31, and 33 showed good inhibitory activities against both JAK2 and HDAC.


Compounds 7, 18, 21, and 30's JAK2 inhibitory action is shown in Figure 1.


Compounds 21 and 30's HDAC and HDAC1 inhibitory activity in Figure 2.

The scientists looked at the drugs' proliferation-inhibitory properties against hematologic malignancy cell lines and solid tumor cell lines based on the JAK and HDAC activities. Investigated were the anti-proliferative properties of representative compounds against three hematologic cancers (HEL, K562 and HL-60). The best combined activity was demonstrated by compounds 21 (IC50 values of 1.87, 2.26, and 0.33 M, respectively) and 30 (IC50 values of 1.22, 3.37, and 0.24 M, respectively). Additionally, compounds 21 and 30 had IC50 values of 0.33 and 0.24 M, respectively, which were both superior than the positive reference fidrotinib (IC50=2.01 M) and comparable to the positive reference SAHA (IC50=0.18 M), both of which were highly effective at inhibiting HEL cells.


Figure 3: Representative drugs' in vitro inhibitory efficacy against solid tumors

In order to confirm the intracellular mechanism of the dual JAK/HDAC inhibitors, the authors et al. used Western blotting. The outcomes shown in Figure 4A and C showed that compounds 21 and 30 significantly increased the expression levels of acetylated H3 and acetylated microtubulin in A549 cells in a dose-dependent manner, indicating that they had a strong blocking effect on the HDAC1/2/3 and HDAC6 signaling pathways while decreasing other signaling The findings showed that the JAK-STAT pathway was severely inhibited, which controls the expression of genes related to cell growth. Their JAK inhibitory activity also outperformed fidrotinib, whereas their HDAC inhibitory activity outperformed SAHA. The scientists examined the inhibitory effects of compounds 21 and 30 on JAK and HDAC in HEL cells in order to research the dual roles of these drugs in hematologic malignancies. The outcomes shown in Figure 4B and D show that compounds 21 and 30 decreased the expression level of p-STAT3Tyr705 while increasing the expression levels of acetyl H3 and acetyl microtubulin in a dose-dependent manner when compared to controls. Their HDAC-inhibitory effects were on par with SAHA's and outperformed fidrotinib's on JAK. The aforementioned findings demonstrated that compounds 21 and 30 dramatically reduced histone deacetylation and STAT3 phosphorylation in solid tumor A549 cells and hematologic malignancy HEL cells by inhibiting HDAC and JAK, indicating that they had dual JAK/HDAC inhibitory effect.

Figure 4 shows the results of a 24-hour treatment with compounds 21 and 30 at 1 and 5 M, respectively, on A549 and HEL cells. Immunoblotting was used to measure the amounts of acetyl H3 (Ac-H3) and acetyl microtubulin (Ac-microtubulin). Compounds 21 and 30 were administered to A549 and (D) HEL cells at concentrations of 1 and 5 M, respectively, and immunoblotting was used to measure the levels of p-STAT3 and STAT3 expression.

The pro-apoptotic activity of compounds 21 and 30 on HEL cells and A549 cells was investigated by the authors et al. by flow cytometry in order to further investigate the anti-tumor action of these substances (Figure 5). According to the findings, compounds 21 and 30 displayed pro-apoptotic effects that were significantly stronger than those of fidrotinib and SAHA, and they also showed some concentration-dependent behavior. Figure 5B depicts the chemicals' pro-apoptotic effects on A549 cells. The findings showed that compounds 21 and 30 were equivalent to SAHA, exhibited a pro-apoptotic activity that was less potent than fidrotinib, and displayed some dose dependence. In summary, chemicals 21 and 30 displayed strong pro-apoptotic activity in HEL cells and weak pro-apoptotic activity in A549 cells.

After 24 hours of action, Figure 5(A) shows how varied doses of chemicals 21 and 30 affect HEL cells' ability to undergo apoptosis. (B) The induction of apoptosis in A549 cells by various doses of compounds 21 and 30 after 36 hours of activity.

The in vivo anticancer activity was then assessed by the authors et al. employing 21 and 30 in the HEL and A549 nude mice transplanted tumor models, respectively. The results are shown in Figure 6, where compounds 21 and 30 significantly decreased the weight and volume of HEL and A549 transplanted tumors. Compound 21 was especially effective, outperforming both compound 30 and the SAHA and fidrotinib combination, while compound 30's inhibitory activity was comparable to that of SAHA and fidrotinib. Compounds 21 and 30 did not significantly affect body weight, as shown in Figure 6C, H, suggesting that they are safe.


Figure 6 In vivo anti-tumor assay. (A) Tumor weight, (B) Tumor volume, (C) Percentage of original body weight, and (D) Photo images. (E) Expression levels of Ac-H3, Ac-tubuin, p-STAT3, and STAT3 in HEL transplanted tumor tissues. (F) Tumor weight, (G) Tumor volume, (H) Percentage of original body weight, and (I) Photo images.